Advances in cell sources of hepatocytes for bioartificial liver

Hangzhou, China

Advances in cell sources of hepatocytes for bioartificial liver

Xiao-Ping Pan and Lan-Juan Li

Hangzhou, China

BACKGROUND:Orthotopic liver transplantation (OLT) is the most effective therapy for liver failure. However, OLT is severely limited by the shortage of liver donors. Bioartificial liver (BAL) shows great potential as an alternative therapy for liver failure. In recent years, progress has been made in BAL regarding genetically engineered cell lines, immortalized human hepatocytes, methods for preserving the phenotype of primary human hepatocytes, and other functional hepatocytes derived from stem cells.

DATA SOURCES:A systematic search of PubMed and ISI Web of Science was performed to identify relevant studies in English language literature using the key words such as liver failure, bioartificial liver, hepatocyte, stem cells, differentiation, and immortalization. More than 200 articles related to the cell sources of hepatocyte in BAL were systematically reviewed.

RESULTS:Methods for preserving the phenotype of primary human hepatocytes have been successfully developed. Many genetically engineered cell lines and immortalized human hepatocytes have also been established. Among these cell lines, the incorporation of BAL with GS-HepG2 cells or alginateencapsulated HepG2 cells could prolong the survival time and improve pathophysiological parameters in an animal model of liver failure. The cBAL111 cells were evaluated using the AMC-BAL bioreactor, which could eliminate ammonia and lidocaine, and produce albumin. Importantly, BAL loading with HepLi-4 cells could significantly improve the blood biochemical parameters, and prolong the survival time in pigs with liver failure. Other functional hepatocytes differentiated from stem cells, such as human liver progenitor cells, have been successfully achieved.

CONCLUSIONS:Aside from genetically modified liver cell lines and immortalized human hepatocytes, other functionalhepatocytes derived from stem cells show great potential as cell sources for BAL. BAL with safe and effective liver cells may be achieved for clinical liver failure in the near future.

(Hepatobiliary Pancreat Dis Int 2012;11:594-605)

bioartificial liver;liver failure; hepatocyte; liver cell source; stem cell; differentiation; immortalization

Introduction

Fulminant liver failure has very high mortality rates approaching 80%.[1,2]To date, orthotopic liver transplantation (OLT) is the most effective therapy for acute liver failure. However, OLT is severely limited by several factors, such as the shortage of liver organ donors, the risk of immune rejection, and the high costs of transplantation. The human liver has a remarkable capacity to regenerate after physical or toxic injury.[3-5]Thus, research has focused on alternative ways to bridge patients with liver failure until successful liver transplantation or the liver of the patient sufficiently regenerates to restore liver function.

Alternative therapies for the treatment of liver failure have been investigated and developed in recent years. The therapies include artificial liver support system,[6-8]bioartificial liver (BAL) support system,[9-11]and hepatocyte transplantation.[12-15]The artificial liver support system is purely a mechanical device or noncell based liver support device, which includes the molecular absorbent and recirculating system (MARS), prometheus system, single pass albumin dialysis (SPAD), selective plasma filtration, and so on. The system could provide detoxification function and improve the survival rate of patients with liver failure.[6-8,16,17]The artificial liver support system promises limited success because of the insufficient replacement of synthetic andmetabolic liver functions and the shortage of normal human plasma supply.[7,18-20]

BAL typically incorporates hepatocytes into bioreactors to provide the important liver functions such as oxidative detoxification, biotransformation, excretion and synthesis. Theoretically, BAL can compensate for the functions of an almost entire liver apart from the secretion of bile. To date, many BAL systems have been applied in clinical trials for liver failure.[21-28]The systems appear to be safe and result in an improvement in the clinical neurologic status and blood biochemical parameters of patients with liver failure. However, none of them has been approved by the medical authorities for routine clinical therapy of liver failure. Nevertheless, some substantial progress has been made in the isolation and preservation of primary human hepatocytes and the establishment of immortalized human hepatocytes and genetically engineered liver cell lines. Stem cells exhibit highly proliferative activity and multipotential ability of differentiation.[29,30]Stem cells are derived from human embryonic stem cells (hESCs),[31-33]human induced pluripotent stem cells (iPSCs),[34,35]human liver progenitor cells (HLPCs),[36]and human mesenchymal stem cells (MSCs),[37]possessing potential ability for differentiation of hepatocytes. They may provide functional hepatocytes for clinical use such as BAL and hepatocyte transplantation.[30,38-40]

Liver cells and bioreactors are the two key components of BAL. The progress of the optimization and modification of bioreactor configurations for BAL has been previously reviewed.[41]In this review, we focus on the advances in liver cell sources, the cellular component of BAL for potential application, including primary porcine hepatocytes, isolation and preservation of primary human hepatocytes, genetically engineered liver cell lines, immortalized human hepatocytes, hESCs, HLPCs, iPSCs, and MSCs.

Primary porcine hepatocytes

Owing to their easy availability and high functional activities, primary porcine hepatocytes are most commonly used in BAL systems in clinical trials. A randomized controlled trial of BAL using primary porcine hepatocytes demonstrated that it is safe and effective in patients with fulminant/subfulminant liver failure.[27]However, primary porcine hepatocytes have a short lifespan and rapidly lose liver functionsin vitro, such as protein synthesis, ureagenesis, and cytochrome P450 activity. As previously reported, several immortalized porcine hepatocytes are established by transfer of SV40 LT into primary porcine hepatocytes.[42-44]This action enables primary porcine hepatocytes to pass and keep their key functions for a long time.

However, porcine endogenous retrovirus (PERV) poses a potential risk in pig-to-human xenozoonosis.[45-47]No evidence ofin vivoproductive infection in humans is currently available.[48-50]Specifically, no PERV infection has been found in long-term immunosuppressed patients who received porcine hepatocyte-based BAL after 8.7 years of follow-up.[51]However, PERV can still be transferred to the membranes of bioreactors, and PERV transmission after short-term direct contact of primary porcine liver cells supernatants with primary human cells could infect the human cells.[52,53]Because of crossspecies immunologic reactions and the potential risk of xenozoonosis, the use of primary porcine hepatocytes is severely hampered in BAL. Thus, the utilization of primary porcine hepatocytes for BAL is prohibited in several European countries.[54]

Isolation and preservation of primary human hepatocytes

Primary human hepatocytes are the most desirable option for BAL. However, liver donors for the isolation of primary human hepatoyctes, as well as for the entire liver transplantation, are scarce. Currently, human livers for hepatocyte isolation are commonly obtained from rejected livers for transplantation or non-transplantable liver organs, such as steatic liver and cirrhotic liver, which possess relatively lower cell yield and viability.[55-58]A standardized and cGMP-conformed method of human hepatocyte isolation has been developed to improve the cell viability for BAL.[59]

Primary human hepatocytes also have limited proliferation and easily lose liver functionin vitro. A number of approaches have been applied to preserve the phenotype of hepatocytes and improve liver-specific functions. Some of these approaches include improvement in cell media components, addition of extracellular matrix,[60,61]modification of biomaterials,[62]three-dimensional culture,[63,64]and co-culture of human hepatocytes and nonparenchymal liver cells[65]for long-time culture of hepatocytein vitro.

Among these methods, co-culture of hepatocytes and nonparenchymal liver cells is very important for preserving liver functions of primary human hepatocytes. Nonparenchymal liver cells include human umbilical vein endothelial cells,[66]endothelial vascular structures,[67]human hepatic stellate cells,[68]human biliary epithelial cells,[69]and bone marrow-derived MSCs.[70]

Importantly, the liver functions of primary human hepatocytes cultured on a bio-modified scaffold or biomaterial membrane are enhanced and maintained at high levels for a longer time, such as modified polyetheretherketone and polyurethane (PEEK-WCPU) membranes and NH3 plasma-grafted PEEK-WCPU membranes.[71,72]These results indicate that biomodified scaffolds are promising biomaterials for the potential use in BAL.

Human hepatoma cell line HepG2 is the most commonly used for BAL because of the advantages of the readily available and importantly secreting human proteins. The liver tumor-derived cell line C3A, a subclone of HepG2, has been used in the ELAD system for clinical trials. Improvement in either survival or biochemical parameters was not observed following the pilot-controlled clinical trial when C3A liver cells were applied to the ELAD system as the cell component of BAL.[21,73]Thus, the liver cell line C3A is of limited use because of its low liver functions.

More importantly, co-culture of HepG2 cells with nonparenchymal liver cells may also improve liver-specific functions.[74-77]Cultivation of C3A cells with alginate beads effectively improves albumin production and easily forms microvilli-like structures characteristic of normal hepatocytes, thereby improving mass transfers and enhancing treatment efficacy.[78,79]The liver differentiation function of FLC-4, a novel hepatoma cell line, can be significantly enhanced when it is cultured in three dimensional cell shape and lactose-silk fibroin conjugate sponges.[80,81]

Genetically engineered tumor liver cell lines

Due to HepG2 cells lacking some key liver functions, some genes such as glutamine synthetase (GS) and hepatocyte nuclear factor (HNF)-4 genes were transferred into HepG2 cells to enhance their weak or lost liver functions. Thus, several novel liver cell lines including HepG2-GS,[82,83]HepG2-GS-3A4,[84,85]HepG2-Bcl2,[86]HepG2/(hArgI+hOTC)4,[87]cBAL119,[88]and HepG2/HNF-4[89]were developed through genetic engineering. The cBAL119 was generated by transduction of HepG2 cells with the human pregnane X receptor (PXR) gene. All these genetically engineered HepG2 can improve the liver-specific functions of hepatocytes for potential application in bioartificial liver. To address intrinsic tumorigenesis, thymidine kinase (TK) was transferred into HepG2 cells (i.e., HepG2/ tk). Moreover, intrasplenic injection of HepG2/tk into rats with liver failure significantly improved blood biochemical parameters and significantly prolonged the survival of rats.[90]These findings indicate that HepG2/ tk is a potential cell source for BAL in the future.

Immortalized human hepatocyte cell lines

To address the issue that primary human hepatocytes easily lose liver functions and do not proliferate for a long-termin vitroculture, several liver cell lines have been developed by gene transfer of the simian virus 40 large antigen gene (SV40 LT) or human telomerase reverse transcriptase such as immortalized human hepatocytes[91-93]and immortalized human fetal hepatocytes.[93-95]

To avoid the risk of SV40 LT tumorigensis, several safer immortalized human hepatocytes have been established, such as the SV40 LT temperature-sensitive cell line[96]and OUMS-29/TK.[97]Moreover, researchers have adopted a reversible immortalization system that uses the Cre-loxP site-specific recombination reaction to remove the targeting SV40 LT gene in reversibly immortalized human hepatocytes, such as NKNT-3, 16-T3, and HepLi4 cells.[98-101]

Immortalized human hepatocytes have been cocultured with human stellate cells or microencapsulated for large-scale culture using roller bottles to improve the differentiation grade and liver functions of immortalized human hepatocytes, which could be candidates for use in BAL.[102,103]

Researchers have already focused on deriving functional hepatocytes from stem cells to address the issue of cell sources of hepatocytes for application in BAL. Stem cells could be differentiated into functional hepatocytes, such as hESCs, HLPCs, iPSCs, and human MSCs.

hESCs

hESCs are isolated from the inner cell mass of blastocysts of fertilized human embryos, having powerful self-renewal and pluripotency.[104,105]hESCs have powerful potential to produce functional hepatocytes as a source of hepatocytes for cellbased replacement therapies and transplantation.[106]Rambhatla et al[107]reported that hESCs could be differentiated into hepatocyte-like cells.

Several protocols for hepatic differentiation of hESCs were developed to provide a source of functional hepatocytes. HNF4α transduction could enhance the efficient generation of functional hepatocytes from hESCs.[108]A relatively homogenous population of hepatocytes was generated from hESCs, which appear to have complete metabolic function of primary hepatocytes.[109]hESCs could be differentiated efficientlyinto functional hepatocytes under a three-stage process, in which hESCs are firstly enabled toward definitive endoderm with activin A and sodium butyrate, followed by dimethyl sulfoxide (DMSO), hepatocyte growth factor, and oncostatin M. hESC-derived functional hepatocytes express several members of cytochrome P450 isozymes, which are capable of converting the substrates to metabolites.[110]Functional hepatocytes could also be generated from hESCs under chemically defined conditions, including a combination of activin, fibroblast growth factor 2, bone morphogenetic protein 4, phosphoinositide 3-kinase inhibition, fibroblast growth factor 10, retinoic acid, and an inhibitor of activin/nodal receptor.[111]

A 3D co-culture system, 3D collagen scaffolds, and ultraweb nanofibers could promote hepatic differentiation of hESCs.[112-116]Specifically, functional hepatocytes differentiated from hESCs could exhibit the potential of liver function of hepatocytes after their transplantation into an animal model.[31]Among these differentiation techniques, the 3D co-culture system of hESCs in combination with tissue engineering may provide a source of functional hepatocytes for potential application in novel BAL.

HLPCs

During the development of human liver, HLPCs found in the early fetal liver bud are termed hepatoblasts, and hepatic oval cells are the bipotent progenitors capable of generating hepatocytes and bile duct cells in adult liver.[117-119]Several human fetal liver progenitor cells from the human fetal liver have been isolated and characterized.[120,121]Several liver progenitor cells were successfully isolated and characterized from normal adult human liver and ischemic liver tissue.[122-124]Adult liver progenitor cells and human fetal hepatic progenitor cells could be differentiated into hepatocyte-like cellsin vitro.[36,122]Importantly, liver progenitor cells could be differentiated into mature hepatocyte-like cells when cultured in a rotary BAL.[125]The HepaRG cell line, a human bipotent liver progenitor cell line, could be differentiated into hepatocyte clusters at a high density with or without DMSO. HepaRG cells are very suitable for BAL application.[126,127]

Human iPSCs

iPSCs are defined as reprogrammed somatic cells that share features of embryonic stem cells, including morphology, expression of key pluripotency genes, and unlimited self-renewal.[128-131]Human iPSCs are generated from somatic cells by retroviral induction of four transcription factors, namely, Oct3/4, Sox2, c-Myc, and Klf4.[130-132]Direct reprogramming of somatic cells is effectively promoted by the maternal transcription factor Glis1 or overexpression of the homeobox gene HEX.[133,134]Human iPSCs could be effectively induced into functional hepatocyte-like cells.[34,35,135]HNF4α transduction and 3D co-culture system could enhance the generation of functional hepatocytes from human iPSCs.[108,112]Mature hepatocyte-like cells could be generated from human iPSCs using an efficient three-step protocol.[136]Hepatocyte-like cells also could be efficiently generated from human ESCs and iPSCs via sequential transduction of SOX17 and HEX.[137]Moreover, the transplantation of mature hepatocyte-like cells derived from human iPSCs into a carbon tetrachloride-injured mouse significantly improves the general condition of a liver-injured mouse after transplantation.[138]

Human MSCs

Human MSCs include human bone marrow-derived MSCs (hBMSCs), human adipose-derived stem cells (hADSCs), human placenta-derived MSCs (hPDMSCs), and so on.[40,139,140]

hBMSCs

Bone marrow is a reservoir of hematopoietic stem cells and MSCs. hBMSCs are suitable as therapeutic tools with relatively easy availability and expansionin vitro. The generation of hepatocyte-like cells from hBMSCs has become a real alternative to the isolation of primary hepatocytes.[141]BMSCs could be effectively induced into functional hepatocytes.[142,143]

Alginate scaffold could enhance the differentiation of BMSCs into hepatocyte-like cells.[144]Hepatic differentiation of hBMSCs could be achieved using tetracycline-regulated HNF3β.[145]A novel 3D biocompatible nanofibrous scaffold could produce functional hepatocyte-like cells derived from hBMSCs.[146-148]The functional hepatocytes derived from hBMSCs could exhibit the functional features of hepatocytes and engraft in the host liver parenchyma of immunocompromised mice.[149]

hADSCs

hADSCs not only share similar characteristics with BMSCs having the potential to differentiate into hepatocytes, but also have a less invasive surgical procedure for harvesting them.[150,151]

Human adipose tissue-derived MSCs gain functional hepatocytes under conditions favoring hepatocyte differentiationin vitro,[37,152,153]which could promote hepatic integrationin vivo.[37]Functional hepatocytes can be produced in large numbers for clinical therapy owing to liver failure.[153]

hADSCs have a similar potential for hepatic differentiation into BMSCs, but have a longer culture period and higher proliferation capacity, indicating that adipose tissue may be an ideal source of large amounts of stem cells for hepatocyte transplantation and BAL.[154]Supplementation of five developmental transcription factors may enhance the differentiation of hADSCs into hepatocytes.[155]hADSCs can differentiate into hepatic lineagein vitro, and hADSCs transplanted into a CCl4-injured SCID mouse model can be differentiated into hepatocytesin vivo.[156]In vitroandin vivostudies also showed that poly-lactide-co-glycolide scaffolds could enhance the hepatogenesis of hADSCs.[157]

hPMSCs

Placenta tissue, a biological waste after delivery, has become a potential source of MSCs for basic and clinical application because of its phenotypic plasticity, easy accessibility, and lack of ethical concerns.[158,159]

Owing to the complex structure of the placenta, the fetal adnexa is composed of the placenta, fetal membranes, and umbilical cord. The cell populations isolated from the placenta consist of human amniotic epithelial cells (hAEC), human amniotic MSCs (hAMSCs), human chorionic MSCs (hCMSCs), and human chorionic trophoblastic cells (hCTC). Amniotic epithelial cells, human amniotic membrane MSCs, and umbilical cord blood MSCs will be the focus.

hAECs as stem cell markers are capable of differentiation toward all the three germ layers. hAECs exhibit hepatocyte-like characteristics and functions, and maintain proliferation.[160,161]hAECs could be differentiated into functional hepatocytes bothin vitroandin vivo,[162]indicating that amniotic epithelial cells are a useful and noncontroversial source of cells for clinical application.

Human amniotic membrane MSCs isolated from the placenta, being immune-privileged and advantageous as therapeutic cells, could be induced into cells of functional hepatocytes under appropriate conditions.[163-166]This finding indicates that amniotic membrane MSCs are a uniquely useful and noncontroversial stem cell source.

hPDMSCs could be manufactured by their isolation and expansion from the human placenta.[167]They also can be expanded for large amounts of hPDMSCs using suspension culture bioreactors. In addition, these stem cells are able to maintain their antigenic phenotype.[168]It was reported that transplantation of human amniotic membrane MSCs could ameliorate carbon tetrachlorideinduced liver cirrhosis in mouse.[169,170]Encouragingly, our team has recently reported that hPDMSCs could not only efficiently differentiate into functional hepatocytes, but also prolong the survival time of ALF pigs through portal vein transplantation.[171]

Human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) can be obtained from the source of cells by different methods.[172-174]hUCB-MSCs can be differentiated into functional hepatocyte-like cells with low immunogenicityin vitroorin vivo.[175-180]

More importantly, hUCB-MSCs can enhance the recovery of CCl4-injured mouse liver, thus they could be used for the treatment of liver dysfunction or liver injury.[181,182]Transplantation of hUCB-MSCs can significantly improve the survival of rats with acute hepatic necrosis or acute liver failure.[183,184]

Ongoing preclinical assessment orin vitrotest

In recent years, genetically engineered cell lines, immortalized human hepatocytes, methods for preserving the phenotype of primary human hepatocytes, and other functional hepatocytes derived from stem cells have been developed for preclinical assessment orin vitrotest.

Modified bioreactors mimicking the liver architecture can support the maintenance of primary human hepatocytes, preserving their liver-specific functions for BAL applications, such as 3D tissue re-structuring bioreactors,[185,186]modified polyetheretherketone and polyethersulfone, and crossed hollow fiber membrane bioreactors.[187]Tissue-like structured bioreactors provide a 3D network of interwoven capillary membranes with integrated oxygenation and decentralized mass exchange.[188,189]

The alginate-encapsulated HepG2 cells or C3A cells in a fluidized bed bioreactor can maintain viability, as well as metabolic, synthetic, and detoxification activities in human plasma after liver failure. Therefore, the system can be improved to form the cellular component of a BAL.[190,191]The BAL system with alginate-encapsulated HepG2 cells could improve systemic parameters of liver failure in a rabbit model, indicating that the encapsulated cells have the potential to develop the BAL for acute liver failure.[192]Moreover, BAL treatment with GS-HepG2 cells could prolong the survival time and improve pathophysiological parameters, such as blood ammonia level and coagulation indices in pigs with ischemic liver failure.[193,194]Another study reported that BAL with FLC-4 cells can prevent irreversible brain damage from hepatic encephalopathy in mini-pigs with acute liver failure.[195]

As previously reported, the immortalized human fetal hepatocyte line cBAL111 could eliminate ammonia, galactose, and lidocaine, and produce albumin when cultured inside the AMC-BAL bioreactor.[196]Importantly, the incorporation of BAL with the reversibly immortalized human hepatocyte line HepLi-4 could improve the Fischer index and serum indirect bilirubin level, and prolong the survival time in pigs with liver failure compared with the liver failure and sham BAL groups.[101]

Human adult liver stem cells which were successfully expanded in a rotary bioreactor perfusion system could be differentiated into mature hepatocyte-like cells. Thus, the rotary BAL incorporating adult liver progenitor cells can be recognized as a potential strategy for BAL.[125]The HepaRG cell line, a liver progenitor cell line, can be differentiated into hepatocyte clusters and it is surrounded by biliary epithelial-like cells at high density after exposure to DMSO, indicating that HepaRG cells are suitable for BAL application.[127]

Problems and prospects

Several cell types as cell sources of BAL have been previously used in clinical trials, including primary porcine hepatocytes,[23,27]primary human hepatocytes,[26]and the liver cell line C3A.[21,73]However, no BAL has been approved by the government for liver failure in clinic. Given the disadvantages of these three cell types, genetically engineered liver cell lines and immortalized human hepatocytes have been successfully used in the animal liver failure model in recent years,[101,194]indicating that they serve as the continuous cell source of metabolically active hepatocytes for BAL.

Aside from genetically engineered liver cells and immortalized human hepatocytes, stem cells (e.g., human ES cells, LPC, and iPS cells) with self-renewal and potential of differentiation into hepatocytes are also promising as sources for BAL. Importantly, mature hepatocyte-like cells from human adult liver stem cells could be expanded in a large quantity for BAL as suitable cell sources.[125]However, a number of problems need to be solved before these cells substitute the presently used cell sources in BAL, including optimization of differentiation protocols.[129,197]

Modified bioreactors mimicking the liver architecture should also be developed to provide a stable long-term culture microenvironment for the improvement of the function of hepatocytes from human liver donors and stem cells. Given the rapid advances in sources of hepatocytes, new-generation BAL with novel and effective liver cells may be achieved for the patients with liver failure in the near future.

Contributors:PXP wrote the article. LLJ proposed and revised the review. LLJ is the guarantor.

Funding:The work was supported by grants from the Chinese High-Tech Research & Development (863) Program (2011AA020104), Science Fund for Creative Research Groups of the National Natural Science Foundation of China (81121002), the Fundamental Research Funds for the Central Universities, and the Technology Group Project for Infectious Disease Control of Zhejiang Province (2009R50041).

Ethical approval:Not needed.

Competing interest:No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

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May 5, 2012

Accepted after revision August 30, 2012

Author Affiliations: State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China (Pan XP and Li LJ)

Lan-Juan Li, MD, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China (Tel/Fax: 86-571-87236759; Email: ljli@zju.edu.cn)

© 2012, Hepatobiliary Pancreat Dis Int. All rights reserved.

10.1016/S1499-3872(12)60230-6