Journal Logo

Reviews

Clinical Application of Pluripotent Stem Cells

An Alternative Cell-Based Therapy for Treating Liver Diseases?

Tolosa, Laia PhD; Pareja, Eugenia MD; Gómez-Lechón, Maria José PhD

Author Information
doi: 10.1097/TP.0000000000001426
  • Free

Liver diseases, either acute liver failure (ALF) or chronic liver failure, also called end-stage liver disease (ESLD),1,2 and life-threatening hepatic metabolic defects, are a major cause of mortality worldwide, and incur one of the highest healthcare burdens.3,4 Although medical therapies are available for early stages of liver diseases, orthotopic liver transplantation (OLT) is the only curative treatment for these irreversible pathologies, but is hindered by the increasing shortage of donor organs. Hence, many patients die while still on the transplanting waiting list.5,6 Despite current medical and surgical therapies, a significant unmet clinical need exists for alternative treatments to support life as a bridge until either a suitable donor liver becomes available or endogenous liver spontaneously regenerates, which might avoid OLT.7

Cell-based therapy is envisaged as a useful therapeutic option to recover and stabilize the lost metabolic function for congenital liver diseases, ALF, ESLD, or for those patients not considered eligible for OLT.8-14

The earliest attempts made in this field involved the allogeneic hepatocyte transplantation (HT).15,16 However, HT are very limited given the scarce availability of suitable donor liver tissue for hepatocyte isolation and, despite the encouraging results obtained, only modest therapeutic benefits of HT have been reported.17-19 Key challenges to implement the use of cell-based liver therapy for large numbers of patients include developing consistent sources of expandable, bankable, engraftable, and functional hepatic cells which can be derived from reproducible methods, thus making them available for transplantation. Stem cells appear to be a promising alternative to primary hepatocytes for cell-based therapies for patients with liver diseases.9,13,14,20-27 Current research on pluripotent stem cells (PSCs), either embryonic stem cells (ESCs) or induced PSC (iPSCs), indicate that these cells are an appealing option to face these challenges.10,12,14,25,26,28 Their capacity for expansion, hepatic differentiation, and self-renewal make them a plausible promising source for generating unlimited numbers of hepatocyte-like cells (HLCs) to treat and repair damaged livers.10,13,14,20,27-29 Replacing hepatocytes with HLCs can be a strategy to not only break away from dependence on organ donation, but to also overcome the shortage of hepatocytes. This is an attractive concept because large numbers of HLCs can be made readily available to any patient on an as-needed basis. The possibility of large-scale pharmaceutical production of HLCs under good manufacturing practice (GMP) conditions is also a guarantee to conduct proper clinical trials to evaluate both safety and efficacy.

This review focuses on the potential use of hPSCs to overcome HT limitations and to provide a real prospect of bringing cell-based therapy for liver diseases in 2 main areas: to make unlimited numbers of HLCs available to extend treatments to many patients and to treat hereditary liver diseases using autologous genetically corrected hiPSC-derived HLCs. These strategies are currently being rigorously tested and validated in preclinical studies before they can be safely transferred to clinical practice with patients.

INDICATIONS OF CELL THERAPY FOR ACUTE AND END-STAGE HEPATIC DISEASES

The shortage of organs for OLT has led transplant centers to extend their criteria to accept marginal donors. Nevertheless, the demand for OLT in human beings far outweighs supply, and mortality among waiting list patients increases. As stated in Table 1, in recent years' hepatic cell therapy has been envisaged as a promising alternative to OLT in these patients. From the clinical viewpoint, transplantation of hepatocytes or HLCs may represent an alternative to OLT in ALF, for correcting genetic disorders that result in metabolically deficient states, for late-stage liver disease such as cirrhosis, or for maintaining liver function in patients who do not meet the clinical eligibility criteria to be candidates for OLT due to advanced age, other diseases or cardiovascular factors. This would imply patient morbidity, improved quality of life, and better survival rates.

TABLE 1
TABLE 1:
Indications and benefits of cell therapy for liver diseases

Nonalcoholic fatty liver disease is currently one of the commonest chronic liver diseases and is estimated to be the most frequent indication for OLT in the next decade.30 However, strategies to enhance resolution of inflammation and fibrosis could also be promising to reverse advanced stages of liver disease. Absence of an effective pharmacological therapy for nonalcoholic fatty liver disease is a major incentive for research into novel therapeutic approaches for this condition.

There are other clinical situations in which patients may suffer liver failure and could benefit from cell therapy. For instance, partial hepatic resection is a feasible and relatively safe procedure and is even used in living-donor liver transplantation as an accepted alternative to cadaveric donor liver transplantation.31 Postoperative liver failure is a dreaded and often fatal complication that sometimes follows partial hepatic resection. In these situations, management principles resemble those applied to patients with ALF or acute-on-chronic liver failure and focus on supporting liver function, but these patients have no indication for OLT. Another large group of patients who could benefit from cell therapy are those with no indications for OLT, such as patients with primary or metastatic malignant hepatic tumors, who require major liver resection after chemotherapy, and who are at high risk of developing postoperative liver failure and, therefore, have no chance of surviving. The more common site of ectopic hepatocyte transplantation is the spleen, especially in patients with major hepatectomy or liver cirrhosis. The spleen can be accessed by direct injection into the splenic artery through a catheter inserted through the femoral artery for implantation in the spleen.19

In all these situations, cell therapy could increase these patients' survival.32 In patients with ESLD, but with a preserved liver function and no indication for OLT, cell therapy could delay disease progression and associated complications, such as the development of hepatocellular carcinoma, and could avoid liver transplant. Finally, the increasing utilization of new therapies in the clinical practice against hepatitis C virus will lead to a significant number of patients with cirrhosis and preserved liver function who do not require OLT. These patients could also benefit from cell therapy.

LIMITATIONS OF HEPATOCYTE TRANSPLANTATION

Clinical demand for hepatocytes cannot be met given the scarcity of current sources of liver tissue for cell isolation, and adult organs being rejected for transplantation, normally of marginal quality, such as severe steatosis, prolonged cold ischemia time and older donors. Despite improvements in hepatocyte isolation methods, it is well known that the mature hepatocytes obtained from these livers often show poor and insufficient functional quality and viability.9,17,18 Cryopreservation and banking allow hepatocytes to be stored for long periods until they are required for both scheduling and emergency treatments, which could thus improve clinical outcomes.18,33-35 However, the detrimental effects of cryopreservation on the viability and metabolic function of adult hepatocytes after thawing are a major concern.18,36,37 Another key issue is the fact that isolated primary adult human hepatocytes rarely, if ever, proliferate in vitro after cellular isolation. This means isolated cells cannot be extended to treat more patients. Although it was initially thought that HT would be less immunogenic than OLT, this has not yet been proved to be the case, and most centers use the same immunosuppressive protocol as they do for OLT.33,34,38 The most significant drawbacks to the clinical application of HT have been extensively reviewed9,17,18,33,39 and are presented in Table 2.

TABLE 2
TABLE 2:
Pros and cons of human hepatic cell sources for cell-based therapy of liver diseases

The clinical application of HT is currently based on disease severity or life quality and has been performed for a variety of indications, including ALF, ESLD, and inborn errors of metabolism to avoid or postpone OLT.11,19,33,38 Several studies have reported that those patients awaiting for OLT, but receiving HT, have a survival advantage compared with those who are not.16,19,34,40-44 Despite encouraging results, HT has had a rather modest and temporary therapeutic effect on transplanted patients to date.9,17,18,38,45 Recently, Hansel et al.19 have extensively reviewed the history of the hepatocyte transplant field, as well as major discoveries and outcomes of the first 100 clinical transplants since 1993.

STEM CELLS AS ALTERNATIVE SOURCES TO HEPATOCYTES FOR CELL-BASED LIVER THERAPY

Stem cells are the main cells of organisms from which all mature body cells derive. Their high proliferative capacity for self-renewal permits their numbers to increase by symmetric division. Stem cells are the source of progenitor cells committed to 1 lineage or to several. Different types of stem cells with hepatic differentiation potential are eligible for generating large numbers of functional HLCs for liver cell therapy.46 These include PSCs and multipotent stem cells, known as adult stem cells (ASCs) (Figure 1). Ideally, these derived cell lines would be highly viable preparations with a robust hepatic function and engraftment capacity, and well characterized. The pros and cons of pluripotent and ASCs are summarized in Table 2.

FIGURE 1
FIGURE 1:
Hepatic cell sources available for cell-based liver therapy. Hepatocytes can be obtained from fetal livers, or from unused neonatal or adult livers, for OLT. Alternative cell sources, which include pluripotent (ESCs and iPSCs) and adult stem cells (AECs, MSCs, HSCs, HPCs), have been proposed for use in cell therapy to treat inborn errors of metabolism, acute liver failure, or chronic liver failure.

Pluripotent Stem Cells

Pluripotent stem cells are characterized by their almost infinite capacity to self-renew indefinitely in culture, while maintaining the potential to generate any cell type in the body. They include both ESCs and iPSCs. In recent years, human PSCs (hPSCs) are envisioned to be the most promising source for cell-based therapies, although the challenges associated with their clinical use are still considerable.

Embryonic Stem Cells

Embryonic stem cells, first isolated in 1998 by Thompson, are PSCs isolated from the inner mass of a blastocyst. Embryology has gained profound insights into key developmental pathways to regulate ESC differentiation, and crucial signals for the hepatic lineage, which include activin A, fibroblastic growth factor, bone morphogenic protein, hepatic growth factor, oncostatin M, and dexamethasone, have been identified.9,47,48 The obtained phenotype seems to come closer to fetal hepatocytes than adult ones,49 yet in vivo, these cells could mature and achieve a more similar phenotype to adult cells.50-54 Human ESCs (hESCs) cells have also been suggested to be resistant to cryopreservation and banking. Several articles have reported the engraftment of ESCs-derived HLCs in livers of various animal models, including transgenic mice, and in mouse models of induced hepatotoxicity.55-57 Despite these encouraging results, the clinical application of hESCs has always been associated with practical and ethical concerns. Therapeutically useful differentiated hESCs must also be safe (ie, nontumorigenic) and need to contribute to liver function in vivo. Nonetheless, the potential of hESCs and their differentiated progeny to generate spontaneous tumors is of particular concern for clinical applications. Good manufacturing practice quality, as defined by both the European Medicines Agency and the Food and Drug Administration, is a requirement for clinical grade cells and would offer optimal safety and quality for cell transplantation.58 This is why hESCs have been derived at the GMP level,9,58-60 and the differentiation protocols that avoid using serum or complex matrices or the addition of other cell types56 are being developed to obtain traceable conditions that can be easily transposable to GMP conditions.

Induced PSCs

They are adult cells that have been genetically reprogrammed to an ESC-like state by being forced to express important genes and factors to maintain the defining properties of ESCs.61,62 Reprogramming somatic cells into iPSCs is a unique opportunity to obtain autologous pluripotent cells for cell therapy, tissue engineering, drug testing, and disease modeling25,63 (Figure 2). In recent years, different strategies have been described to reprogram cells using alternatives to integrative viral-mediated strategies and to use other cell types susceptible to reprogramming, such as blood cells or mesenchymal stem cells (MSCs).64 Several protocols to differentiate iPSCs into HLCs have been defined (25,63,65-68 for a review, see Okano et al and Gerbal-Chaloin et al25,63), and despite them still differing from adult hepatocytes,49 they can provide a limitless supply of HLCs. On top of this, the latest advances made in gene editing technologies have vigorously endorsed the possibility of correcting the genetic anomalies that induce the disease, and of obtaining disease-free autologous cells from patient-specific iPSCs. The differentiated derivatives from corrected patient hiPSCs have the potential to be employed in autologous cell therapy.14,25,26 Personalized cell therapy using hiPSCs would likely avoid rejection, and thus immunosuppression. However, there is still some controversy about the immunogenicity of these cells, which should be assessed before these autologous cells are clinically used.69,70 Examples of using iPSC-derived cells for tissue repair in animal models of metabolopathies or liver injury have been reported.28,71-73 In light of these studies, transplantation of the HLCs that derive from autologous genetically corrected hiPSCs shows great promise for treating hereditary liver diseases. The development of hiPSC-based clinical trials will have to consider an essential step of mutation detection, although the guidelines and acceptability criteria have not yet been established.64 In line with this, the pioneering hiPSC clinical trial for the treatment of macular degeneration in Japan has been stopped because one of the hiPSCs did not pass the genomic validation step.64 As with ESCs, production under GMP conditions should also be considered.

FIGURE 2
FIGURE 2:
Applications of iPSCs generated from patients. Reprogramming somatic cells into iPSCs to obtain derived HLCs could be used in autologous hepatic cell therapy, tissue engineering, drug testing, and disease modeling.

Adult Stem Cells

Adult stem cells are multipotent stem cells with a relatively limited differentiation potential that reside together with specialized cell types of adult tissues. Indeed, distinct populations of ASC have been envisaged to be used for cell therapy.

Hematopoietic Stem Cells

One of the most studied stem cell sources for liver cell transplant is hematopoietic stem cells (HSCs), responsible for the renewal of blood cells, which can be isolated from peripheral blood, bone marrow. or umbilical cord blood.74 The transdifferentiation of human HSCs (hHSCs) into hepatocytes has been studied and their ability to express albumin and CK18 when cocultured with damaged liver tissue has been reported.75 The functionality of these cells has been proven by the transplantation of hepatocytes into recipient mice with liver failure induced by carbon tetrachloride.76 However, most studies have focused on using undifferentiated HSCs, and many trials have obtained promising results when undifferentiated HSCs were transplanted mainly into patients with liver cirrhosis.77-80 Despite these encouraging results, there are still many open questions on the homing processes and mechanisms by which these cells act in the liver. Most clinical trial results have shown only temporary effects, which suggests that HSCs act by conferring trophic support rather than through transdifferentiation.79

Mesenchymal Stem Cells

Mesenchymal stem cells are multipotent cells present in different tissues, such as bone marrow, cord blood, adipose tissue, and amniotic fluid, which can replicate as undifferentiated cells. They have the potential to differentiate into not only lineages of mesenchymal tissues,22,29 but also into tissue cells that derive from other embryonic layers, including HLCs.81-83 In the last decade, several studies have reported the plasticity of MSCs toward a hepatocyte-like phenotype.22,84-87 Intensive research is being conducted to evaluate the efficacy of hMSCs as an alternative cell source to HT to treat liver diseases. One key advantage of hMSCs is their immunological properties, which render them less immunogenic and possibly able to induce tolerance, as highlighted by promising in vitro studies and clinical trials.24,88,89 To date, however, all studies have demonstrated that MSCs exert their effects through paracrine signaling and the secretion of factors to influence immunological responses.23

Hepatic Progenitor Cells

Hepatic progenitor cells (HPCs) are bipotential cells able to give rise to both biliary epithelia and hepatocytes which, when normal liver regeneration is impaired, become active and replace damaged cells.90 Hepatic progenitor cells isolated from human fetal liver are less immunogenic, highly propagative, and more challenging for cryopreservation than adult ones.21 Although HPCs can be induced to differentiate into cells with the morphological, phenotypic, and functional characteristics of mature hepatocytes,91 their use without differentiation has been proposed. The Promethera program presently uses liver-derived progenitor cells expanded in vitro which are injected into patients with various hereditary metabolic diseases.8,12 However, the clinical outcome of the first transplantation in a 3-year-old girl with OTC deficiency has not yet been reported.92 The use of EpCAM+ cells to treat different liver diseases has been assessed at the Liver Institute in Hyderabad, India. An early publication revealed that 25 patients with liver cirrhosis who were infused with human fetal liver-derived stem cells (EpCAM+) obtained improved mean MELD scores.93 However, details of these patients' long-term outcomes are still not available, and further information is needed to elucidate the potential efficacy of this strategy.23

Amnion Epithelial Cells

Human amnion epithelial cells are isolated from term placenta and have been described to have similar surface markers and gene expression profiles to those reported for hESCs.94 They can be differentiated into an hepatic phenotype,95-97 and preclinical studies that used undifferentiated cells in animal models of metabolic liver disease and ALF have provided encouraging results.98 Because human amnion epithelial cell are isolated from tissue that is normally discarded after birth, they are quite plentiful and easily isolated, do not produce tumors when transplanted, and could prove to be a uniquely useful and noncontroversial stem cell source.98

POTENTIAL BENEFITS OF PLURIPOTENT STEM CELL-BASED LIVER THERAPY

Cell therapy in hepatology offers numerous potential advantages compared with OLT, is less invasive, involves fewer risks of morbidity and mortality, is less expensive, and does not contraindicate or preclude OLT over time. To overcome the recognized limitations of HT, the use of alternative stem cell-derived sources has been recently proposed, but hPSCs have gained a reputation for hepatic cell-based therapy in the last few years. Thus, the development of hPSC-based therapies for treating liver diseases, including inborn liver metabolic diseases, is currently being investigated worldwide to potentially produce a more sustained and significant metabolic support of the liver. Moreover, the HLCs that derive from hPSCs are emerging cell-based systems that could provide a stable source of hepatocytes for multiple applications, including the drug safety screening of new drugs,99-102 disease modeling,103,104 and hepatitis C virus replication.105,106

These cells can be: (1) expanded in vitro, which would abolish the limit of organ shortage; (2) differentiated toward hepatic lineage; (3) cryopreserved so they are constantly available, and extensive quality testing can be performed; (4) banked, thus allowing cells to be stored for long periods for future use in both scheduling and emergency treatments with any patient in need; (5) transplanted many times; and (6) infused without major surgery.

The progress made in recent years with hiPSCs has opened up many potential gateways for therapeutics. Disease-free autologous hiPSCs, generated through ex vivo reprogramming, can undergo genetic manipulation to correct inborn metabolic defects and then be differentiated into hHLCs; hereditary liver disease treatment can be taken a step further by combining genetic correction technology with autologous cell transplantation (patient-specific therapies), which would likely avoid the risk of rejection and the need for lifelong immunosuppression.10,12-14,25,26,28

IMMUNOGENICITY AND TUMORIGENICITY OF PLURIPOTENT STEM CELLS AND THEIR DERIVED HLCS

Immunogenicity of hPSCs remains the major bottleneck for successful clinical applications. A high tumor immune surveillance would prevent tumor formation by eliminating cancerous and/or precancerous cells before they can cause harm. However, reduced immunogenicity would not only facilitate cell engraftment but also tumor formation.

Two categories of hPSCs grafts exist with different immunogenic properties: autologous and allogeneic cell transplantations. One key challenge is the immune rejection of the cells derived from allogeneic hESCs by recipients. These cells have been reported to evade immune destruction given a low immunostimulatory potential. Although the major histocompatibility complex (MHC)-I levels in hESCs are sufficient for rejection by cytotoxic T cells, it has been suggested that the immunostimulatory capacity of cells is very poor. Thus, immunosuppressive regimes for hESC-based therapeutics could be greatly reduced compared with conventional organ transplantation because the direct allorejection processes of hESCs and their derivatives are considerably weaker.107,108 The challenge of immune rejection has been speculated to be reduced by the recent discovery of iPSCs, which could become a renewable source of autologous cells for cell therapy. This expectation is based on the hope that patient-specific hiPSCs differentiated into autologous cells for transplantation into the same patient entails no immune rejection concern.109,110 However, hiPSCs display several genetic and epigenetic abnormalities that could promote tumorigenicity and immunogenicity in vivo.111-113

The differential immune recognition between differentiated and undifferentiated hPSCs has also been investigated. Undifferentiated, but not differentiated, PSCs have been reported to possess immune privilege properties.114 Work with ESCs has shown variability in MHC expression and increased immunogenicity after differentiation.114 Yet immune rejection accelerates when MHC molecules are upregulated during ESCs differentiation.115,116 Early studies have also demonstrated that both undifferentiated hESCs and iPSCs show a lower expression of MHC class I, and the complete absence of MHC class II antigens and co-stimulatory molecules (CD80 and CD86), compared to their differentiated progeny.115,117 On top of this, it has been suggested that epigenetic mechanisms regulate MHC expression during differentiation. Hypermethylation of the MHC class II gene in undifferentiated hESCs and iPSCs has been found to be a major epigenetic repression mechanism in gene expression.118 These findings suggest that undifferentiated cells would be less susceptible to immune recognition than their derived differentiated cell types. However, a recent report has investigated the immunogenicity of several hESC and hiPSC lines and their hepatic derivatives. Its results suggested that the derivatives of both types of cell lines would likely induce an immune response, although immune privilege characteristics could occur through lower expression levels of MHC, antigen-presenting cells, and the modulation of immune privilege genes.72

Recent studies, conducted with a humanized mice model reconstituted with a functional human immune system, have shown that the local expression of CTLA4-Ig and PD-L1 could effectively confer immune protection to hESC-derivatives.108 Further in vivo studies will be the next step to determine to what extent appropriately and terminally differentiated stem cell lineages will induce the immunoresponse after transplantation.

Tumorigenicity is another potential hurdle that could hinder large-scale hESC and hiPSC clinical implementation. The intrinsic qualities of self-renewal and pluripotency that make PSCs so therapeutically promising are also responsible for an equally fundamental tumorigenic potential. Pluripotent stem cell tumorigenicity can be ultimately divided into 2 separate categories, malignant transformation of differentiated PSCs and benign teratoma formation from residual undifferentiated PSCs, either of which can produce tumors that consist of either 1 or all 3 germ layers, respectively.119 The potential risk of PSC tumorigenicity in humans has been evaluated in the last few years by small and large animal studies that utilized ESC-based and iPSC-based therapies.120,121 Initial studies have shown that ESCs injected into mice in their undifferentiated state result in teratoma formation, which kills animals,122 whereas no teratomas are produced when differentiated cells are injected. Some reports have indicated tumor formation after the transplantation of ESC-derived hepatic cells despite predifferentiation120,123 and transplanted cells that contained a number of undifferentiated ESCs.124 Other reports have shown that the transplantation of highly differentiated cells does not entail tumor development,125 which suggests that steering ESCs to an appropriate state could be an important step for safe and effective cell therapies. The induction of pluripotency itself by reprogramming somatic cells has also been linked to tumorigenic transformation by creating genomic aberrations at chromosomal and subchromosomal levels. Numerous deletions of tumor-suppressor genes in iPSCs have been found immediately after pluripotency induction, which were absent from the somatic cells of origin.126 A recent analysis of the tumorigenic genes of several iPSCs lines has revealed 593 iPSC consensus genes, of which almost half were also expressed in human tumor cell lines and cancer tissues. Moreover, notably 5 oncogenes were overexpressed in iPSCs and the oncogene RAB25, a RAS oncogene family member, in differentiated cell derivatives, which suggests that these iPSC consensus genes may entail the risk of tumorigenesis and cancers.113 Ultimately, genetic modifications have profound functional implications and promote tumorigenic qualities, such as increased proliferation, growth factor independence, and higher frequencies of tumor-initiating cells. The effect of culture adaption is more noticeable in the accumulation of gross chromosomal abnormalities in high passage PSCs. The fact that such genetic lesions are generated stochastically suggests that significant variations may arise between laboratories and even between subclones from the same cell lines. The prospective removal (eg, removal before transplantation) of tumorigenic cells has been proposed, which would provide the highest level of patient safety and is optimally achieved through utilizing intrinsic cell properties, such as surface antigens.119 In addition to removing residual undifferentiated cells, attention should also be paid to ensure the removal of genetically abnormal cells, irrespectively of their differentiation status. In summary, well-defined methods to reduce the expression of oncogenic genes in iPSCs, including protocols for their complete and safe hepatic differentiation, should be established to minimize the tumorigenicity of transplanted cells.28,114

CONCLUSIONS AND FUTURE PERSPECTIVES

There is a therapeutic need for cell-based approaches to treat liver diseases to overcome the growing discrepancy between demand for, and availability of, donor livers for OLT. Approaches using hepatocytes are already available in clinical practice, and the preclinical proof-of-concept exists that hPSC-derived hepatocytes can improve disease in animal models. The emerging field of hiPSC research is continually evolving, and the ethical and immune issues related with the use of hESCs are being circumvented. These cell therapies are amenable to gene modification (eg, to correct congenital defects) and to scalability of production. In the present day, the long-term safety, tolerability, and efficacy of these cell-based treatments are key issues to be addressed before hPSCs can be achieved to treat liver diseases. Therefore, current challenges include the development of reliable and efficient processes to differentiate hPSCs into mature HLCs, avoiding the use of viral vectors or changes in cell cycle regulators so as to evade tumorigenicity concerns, and large-scale production and banking of high-quality HLCs for transplantation. In addition, the critical issues still not solved to accelerate the translation of cell therapy to clinical practice are preconditioning treatments of the recipient liver to enhance the engraftment and proliferation of donor cells, development of noninvasive, and accurate tracking or monitoring methods for cell survival and homing after transplantation, and optimization of immunosuppression protocols for transplant recipients. Finally, it is paramount that preclinical testing in large animal models before the clinical application in well-designed clinical trials fully establishes the safety profile of such therapies and defines target patient groups.

REFERENCES

1. Jalan R, Gines P, Olson JC, et al. Acute-on chronic liver failure. J Hepatol. 2012;57:1336–1348.
2. Polson J, Lee WM, American Association for the Study of Liver D. AASLD position paper: the management of acute liver failure. Hepatology. 2005;41:1179–1197.
3. Blachier M, Leleu H, Peck-Radosavljevic M, et al. The burden of liver disease in Europe: a review of available epidemiological data. J Hepatol. 2013;58:593–608.
4. Williams R. Global challenges in liver disease. Hepatology. 2006;44:521–526.
5. Kemmer N, Alsina A, Neff GW. Orthotopic liver transplantation in a multiethnic population: role of spatial accessibility. Transplant Proc. 2011;43:3780–3782.
6. Kim WR, Therneau TM, Benson JT, et al. Deaths on the liver transplant waiting list: an analysis of competing risks. Hepatology. 2006;43:345–351.
7. Viswanathan P, Gupta S. New directions for cell-based therapies in acute liver failure. J Hepatol. 2012;57:913–915.
8. Cantz T, Sharma AD, Ott M. Concise review: cell therapies for hereditary metabolic liver diseases-concepts, clinical results, and future developments. Stem Cells. 2015;33:1055–1062.
9. Forbes SJ, Gupta S, Dhawan A. Cell therapy for liver disease: from liver transplantation to cell factory. J Hepatol. 2015;62(1 Suppl):S157–S169.
10. Huebert RC, Rakela J. Cellular therapy for liver disease. Mayo Clin Proc. 2014;89:414–424.
11. Jorns C, Ellis EC, Nowak G, et al. Hepatocyte transplantation for inherited metabolic diseases of the liver. J Intern Med. 2012;272:201–223.
12. Kadyk LC, Collins LR, Littman NJ, et al. Proceedings: moving toward cell-based therapies for liver disease. Stem Cells Transl Med. 2015;4:207–210.
13. Nicolas C, Wang Y, Luebke-Wheeler J, et al. Stem cell therapies for treatment of liver disease. Biomedicines. 2016;4:2.
14. Yu Y, Wang X, Nyberg SL. Potential and challenges of induced pluripotent stem cells in liver diseases treatment. J Clin Med. 2014;3:997–1017.
15. Fox IJ, Roy-Chowdhury J. Hepatocyte transplantation. J Hepatol. 2004;40:878–886.
16. Strom SC, Fisher RA, Thompson MT, et al. Hepatocyte transplantation as a bridge to orthotopic liver transplantation in terminal liver failure. Transplantation. 1997;63:559–569.
17. Dhawan A. Clinical human hepatocyte transplantation: current status and challenges. Liver Transpl. 2015;21(Suppl 1):S39–S44.
18. Gramignoli R, Vosough M, Kannisto K, et al. Clinical hepatocyte transplantation: practical limits and possible solutions. Eur Surg Res. 2015;54:162–177.
19. Hansel MC, Gramignoli R, Skvorak KJ, et al. The history and use of human hepatocytes for the treatment of liver diseases: the first 100 patients. Curr Protoc Toxicol. 2014;62:14.12.1–14.12.23.
20. Esrefoglu M. Role of stem cells in repair of liver injury: experimental and clinical benefit of transferred stem cells on liver failure. World J Gastroenterol. 2013;19:6757–6773.
21. Habeeb MA, Vishwakarma SK, Bardia A, et al. Hepatic stem cells: a viable approach for the treatment of liver cirrhosis. World J Stem Cells. 2015;7:859–865.
22. Hu C, Li L. In vitro and in vivo hepatic differentiation of adult somatic stem cells and extraembryonic stem cells for treating end stage liver diseases. Stem Cells Int. 2015;2015:871972.
23. Lanzoni G, Oikawa T, Wang Y, et al. Concise review: clinical programs of stem cell therapies for liver and pancreas. Stem Cells. 2013;31:2047–2060.
24. Lysy PA, Campard D, Smets F, et al. Persistence of a chimerical phenotype after hepatocyte differentiation of human bone marrow mesenchymal stem cells. Cell Prolif. 2008;41:36–58.
25. Okano H, Nakamura M, Yoshida K, et al. Steps toward safe cell therapy using induced pluripotent stem cells. Circ Res. 2013;112:523–533.
26. Singh VK, Kalsan M, Kumar N, et al. Induced pluripotent stem cells: applications in regenerative medicine, disease modeling, and drug discovery. Front Cell Dev Biol. 2015;3:2.
27. Zhang Z, Wang FS. Stem cell therapies for liver failure and cirrhosis. J Hepatol. 2013;59:183–185.
28. Harding J, Mirochnitchenko O. Preclinical studies for induced pluripotent stem cell-based therapeutics. J Biol Chem. 2014;289:4585–4593.
29. Allameh A, Kazemnejad S. Safety evaluation of stem cells used for clinical cell therapy in chronic liver diseases; with emphasize on biochemical markers. Clin Biochem. 2012;45:385–396.
30. Agopian VG, Kaldas FM, Hong JC, et al. Liver transplantation for nonalcoholic steatohepatitis: the new epidemic. Ann Surg. 2012;256:624–633.
31. Broelsch CE, Whitington PF, Emond JC, et al. Liver transplantation in children from living related donors. Surgical techniques and results. Ann Surg. 1991;214:428–437.
32. Balzan S, Belghiti J, Farges O, et al. The “50-50 criteria” on postoperative day 5: an accurate predictor of liver failure and death after hepatectomy. Ann Surg. 2005;242:824–828.
33. Puppi J, Strom SC, Hughes RD, et al. Improving the techniques for human hepatocyte transplantation: report from a consensus meeting in London. Cell Transplant. 2012;21:1–10.
34. Ribes-Koninckx C, Pareja Ibars E, Agrasot MA, et al. Clinical outcome of hepatocyte transplantation in four pediatric patients with inherited metabolic diseases. Cell Transplant. 2012.
35. Stéphenne X, Najimi M, Sokal EM. Hepatocyte cryopreservation: is it time to change the strategy? World J Gastroenterol. 2010;16:1–14.
36. Terry C, Dhawan A, Mitry RR, et al. Optimization of the cryopreservation and thawing protocol for human hepatocytes for use in cell transplantation. Liver Transpl. 2010;16:229–237.
37. Tolosa L, Bonora-Centelles A, Donato MT, et al. Influence of platelet lysate on the recovery and metabolic performance of cryopreserved human hepatocytes upon thawing. Transplantation. 2011;91:1340–1346.
38. Dhawan A, Puppi J, Hughes RD, et al. Human hepatocyte transplantation: current experience and future challenges. Nat Rev Gastroenterol Hepatol. 2010;7:288–298.
39. Hughes RD, Mitry RR, Dhawan A. Current status of hepatocyte transplantation. Transplantation. 2012;93:342–347.
40. Bilir BM, Guinette D, Karrer F, et al. Hepatocyte transplantation in acute liver failure. Liver Transpl. 2000;6:32–40.
41. Fisher RA, Bu D, Thompson M, et al. Defining hepatocellular chimerism in a liver failure patient bridged with hepatocyte infusion. Transplantation. 2000;69:303–307.
42. Habibullah CM, Syed IH, Qamar A, et al. Human fetal hepatocyte transplantation in patients with fulminant hepatic failure. Transplantation. 1994;58:951–952.
43. Pareja E, Gomez-Lechon MJ, Cortes M, et al. Human hepatocyte transplantation in patients with hepatic failure awaiting a graft. Eur Surg Res. 2013;50:273–281.
44. Pietrosi G, Vizzini GB, Gruttadauria S, et al. Clinical applications of hepatocyte transplantation. World J Gastroenterol. 2009;15:2074–2077.
45. Allen KJ, Mifsud NA, Williamson R, et al. Cell-mediated rejection results in allograft loss after liver cell transplantation. Liver Transpl. 2008;14:688–694.
46. Godoy P, Schmidt-Heck W, Natarajan K, et al. Gene networks and transcription factor motifs defining the differentiation of stem cells into hepatocyte-like cells. J Hepatol. 2015;63:934–942.
47. Agarwal S, Holton KL, Lanza R. Efficient differentiation of functional hepatocytes from human embryonic stem cells. Stem Cells. 2008;26:1117–1127.
48. Cai J, Zhao Y, Liu Y, et al. Directed differentiation of human embryonic stem cells into functional hepatic cells. Hepatology. 2007;45:1229–1239.
49. Baxter M, Withey S, Harrison S, et al. Phenotypic and functional analyses show stem cell-derived hepatocyte-like cells better mimic fetal rather than adult hepatocytes. J Hepatol. 2015;62:581–589.
50. Cameron K, Tan R, Schmidt-Heck W, et al. Recombinant laminins drive the differentiation and self-organization of hESC-derived hepatocytes. Stem Cell Reports. 2015;5:1250–1262.
51. Duan Y, Catana A, Meng Y, et al. Differentiation and enrichment of hepatocyte-like cells from human embryonic stem cells in vitro and in vivo. Stem Cells. 2007;25:3058–3068.
52. Hay DC, Fletcher J, Payne C, et al. Highly efficient differentiation of hESCs to functional hepatic endoderm requires activinA and Wnt3a signaling. Proc Natl Acad Sci U S A. 2008;105:12301–12306.
53. Lavon N, Yanuka O, Benvenisty N. Differentiation and isolation of hepatic-like cells from human embryonic stem cells. Differentiation. 2004;72:230–238.
54. Rashidi H, Alhaque S, Szkolnicka D, et al. Fluid shear stress modulation of hepatocyte-like cell function. Arch Toxicol. 2016;90:1757–1761.
55. Basma H, Soto-Gutierrez A, Yannam GR, et al. Differentiation and transplantation of human embryonic stem cell-derived hepatocytes. Gastroenterology. 2009;136:990–999.
56. Tolosa L, Caron J, Hannoun Z, et al. Transplantation of hESC-derived hepatocytes protects mice from liver injury. Stem Cell Res Ther. 2015;6:246.
57. Woo DH, Kim SK, Lim HJ, et al. Direct and indirect contribution of human embryonic stem cell-derived hepatocyte-like cells to liver repair in mice. Gastroenterology. 2012;142:602–611.
58. Unger C, Skottman H, Blomberg P, et al. Good manufacturing practice and clinical-grade human embryonic stem cell lines. Hum Mol Genet. 2008;17(R1):R48–R53.
59. Canham MA, Van Deusen A, Brison DR, et al. The molecular karyotype of 25 clinical-grade human embryonic stem cell lines. Sci Rep. 2015;5:17258.
60. Carpenter MK, Rao MS. Concise review: making and using clinically compliant pluripotent stem cell lines. Stem Cells Transl Med. 2015;4:381–388.
61. Takahashi K, Tanabe K, Ohnuki M, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007;131:861–872.
62. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;126:663–676.
63. Gerbal-Chaloin S, Funakoshi N, Caillaud A, et al. Human induced pluripotent stem cells in hepatology: beyond the proof of concept. Am J Pathol. 2014;184:332–347.
64. Hannoun Z, Steichen C, Dianat N, et al. The potential of induced pluripotent stem cell derived hepatocytes. J Hepatol. 2016;65:82–99.
65. Kondo Y, Iwao T, Nakamura K, et al. An efficient method for differentiation of human induced pluripotent stem cells into hepatocyte-like cells retaining drug metabolizing activity. Drug Metab Pharmacokinet. 2014;29:237–243.
66. Si-Tayeb K, Noto FK, Nagaoka M, et al. Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells. Hepatology. 2010;51:297–305.
67. Song Z, Cai J, Liu Y, et al. Efficient generation of hepatocyte-like cells from human induced pluripotent stem cells. Cell Res. 2009;19:1233–1242.
68. Sullivan GJ, Hay DC, Park IH, et al. Generation of functional human hepatic endoderm from human induced pluripotent stem cells. Hepatology. 2010;51:329–335.
69. Araki R, Uda M, Hoki Y, et al. Negligible immunogenicity of terminally differentiated cells derived from induced pluripotent or embryonic stem cells. Nature. 2013;494:100–104.
70. Zhao T, Zhang ZN, Rong Z, et al. Immunogenicity of induced pluripotent stem cells. Nature. 2011;474:212–215.
71. Asgari S, Moslem M, Bagheri-Lankarani K, et al. Differentiation and transplantation of human induced pluripotent stem cell-derived hepatocyte-like cells. Stem Cell Rev. 2013;9:493–504.
72. Chen HF, Yu CY, Chen MJ, et al. Characteristic expression of major histocompatibility complex and immune privilege genes in human pluripotent stem cells and their derivatives. Cell Transplant. 2015;24:845–864.
73. Yusa K, Rashid ST, Strick-Marchand H, et al. Targeted gene correction of alpha1-antitrypsin deficiency in induced pluripotent stem cells. Nature. 2011;478:391–394.
74. Austin TW, Lagasse E. Hepatic regeneration from hematopoietic stem cells. Mech Dev. 2003;120:131–135.
75. Shu SN, Wei L, Wang JH, et al. Hepatic differentiation capability of rat bone marrow-derived mesenchymal stem cells and hematopoietic stem cells. World J Gastroenterol. 2004;10:2818–2822.
76. Jang YY, Collector MI, Baylin SB, et al. Hematopoietic stem cells convert into liver cells within days without fusion. Nat Cell Biol. 2004;6:532–539.
77. Burganova GR. Effectiveness of autologous hematopoietic stem cells transplantation in patients with liver cirrhosis. Eksp Klin Gastroenterol. 2012:91–97.
78. King A, Barton D, Beard HA, et al. REpeated AutoLogous Infusions of STem cells In Cirrhosis (REALISTIC): a multicentre, phase II, open-label, randomised controlled trial of repeated autologous infusions of granulocyte colony-stimulating factor (GCSF) mobilised CD133+ bone marrow stem cells in patients with cirrhosis. A study protocol for a randomised controlled trial. BMJ Open. 2015; 5:e007700.
79. Margini C, Vukotic R, Brodosi L, et al. Bone marrow derived stem cells for the treatment of end-stage liver disease. World J Gastroenterol. 2014;20:9098–9105.
80. Zekri AR, Salama H, Medhat E, et al. The impact of repeated autologous infusion of haematopoietic stem cells in patients with liver insufficiency. Stem Ce ll Res Ther. 2015;6:118.
81. Lee RH, Kim B, Choi I, et al. Characterization and expression analysis of mesenchymal stem cells from human bone marrow and adipose tissue. Cell Physiol Biochem. 2004;14:311–324.
82. Pittenger MF, Martin BJ. Mesenchymal stem cells and their potential as cardiac therapeutics. Circ Res. 2004;95:9–20.
83. Sgodda M, Aurich H, Kleist S, et al. Hepatocyte differentiation of mesenchymal stem cells from rat peritoneal adipose tissue in vitro and in vivo. Exp Cell Res. 2007;313:2875–2886.
84. Aurich H, Sgodda M, Kaltwasser P, et al. Hepatocyte differentiation of mesenchymal stem cells from human adipose tissue in vitro promotes hepatic integration in vivo. Gut. 2009;58:570–581.
85. Banas A, Teratani T, Yamamoto Y, et al. Adipose tissue-derived mesenchymal stem cells as a source of human hepatocytes. Hepatology. 2007;46:219–228.
86. Bonora-Centelles A, Jover R, Mirabet V, et al. Sequential hepatogenic transdifferentiation of adipose tissue-derived stem cells: relevance of different extracellular signaling molecules, transcription factors involved, and expression of new key marker genes. Cell Transplant. 2009;18:1319–1340.
87. Seo MJ, Suh SY, Bae YC, et al. Differentiation of human adipose stromal cells into hepatic lineage in vitro and in vivo. Biochem Biophys Res Commun. 2005;328:258–264.
88. Chen Y, Xiang LX, Shao JZ, et al. Recruitment of endogenous bone marrow mesenchymal stem cells towards injured liver. J Cell Mol Med. 2010;14(6B):1494–1508.
89. Squillaro T, Peluso G, Galderisi U. Clinical trials with mesenchymal stem cells: an update. Cell Transplant. 2015;25:829–848.
90. Fausto N, Campbell JS. The role of hepatocytes and oval cells in liver regeneration and repopulation. Mech Dev. 2003;120:117–130.
91. He Z, Feng M. Activation, isolation, identification and culture of hepatic stem cells from porcine liver tissues. Cell Prolif. 2011;44:558–566.
92. Sokal EM, Stephenne X, Ottolenghi C, et al. Liver engraftment and repopulation by in vitro expanded adult derived human liver stem cells in a child with ornithine carbamoyltransferase deficiency. JIMD Rep. 2014;13:65–72.
93. Khan AA, Shaik MV, Parveen N, et al. Human fetal liver-derived stem cell transplantation as supportive modality in the management of end-stage decompensated liver cirrhosis. Cell Transplant. 2010;19:409–418.
94. Miki T, Lehmann T, Cai H, et al. Stem cell characteristics of amniotic epithelial cells. Stem Cells. 2005;23:1549–1559.
95. Marongiu F, Gramignoli R, Dorko K, et al. Hepatic differentiation of amniotic epithelial cells. Hepatology. 2011;53:1719–1729.
96. Miki T, Marongiu F, Ellis EC, et al. Production of hepatocyte-like cells from human amnion. Methods Mol Biol. 2009;481:155–168.
97. Vaghjiani V, Vaithilingam V, Saraswati I, et al. Hepatocyte-like cells derived from human amniotic epithelial cells can be encapsulated without loss of viability or function in vitro. Stem Cells Dev. 2014;23:866–876.
98. Strom SC, Skvorak K, Gramignoli R, et al. Translation of amnion stem cells to the clinic. Stem Cells Dev. 2013;22(Suppl 1):96–102.
99. Medine CN, Lucendo-Villarin B, Storck C, et al. Developing high-fidelity hepatotoxicity models from pluripotent stem cells. Stem Cells Transl Med. 2013;2:505–509.
100. Szkolnicka D, Farnworth SL, Lucendo-Villarin B, et al. Accurate prediction of drug-induced liver injury using stem cell-derived populations. Stem Cells Transl Med. 2014;3:141–148.
101. Szkolnicka D, Lucendo-Villarin B, Moore JK, et al. Reducing hepatocyte injury and necrosis in response to paracetamol using noncoding RNAs. Stem Cells Transl Med. 2016;5:764–772.
102. Holmgren G, Sjogren AK, Barragan I, et al. Long-term chronic toxicity testing using human pluripotent stem cell-derived hepatocytes. Drug Metab Dispos. 2014;42:1401–1406.
103. Cayo MA, Cai J, DeLaForest A, et al. JD induced pluripotent stem cell-derived hepatocytes faithfully recapitulate the pathophysiology of familial hypercholesterolemia. Hepatology. 2012;56:2163–2171.
104. Rashid ST, Corbineau S, Hannan N, et al. Modeling inherited metabolic disorders of the liver using human induced pluripotent stem cells. J Clin Invest. 2010;120:3127–3136.
105. Roelandt P, Obeid S, Paeshuyse J, et al. Human pluripotent stem cell-derived hepatocytes support complete replication of hepatitis C virus. J Hepatol. 2012;57:246–251.
106. Sa-Ngiamsuntorn K, Wongkajornsilp A, Phanthong P, et al. A robust model of natural hepatitis C infection using hepatocyte-like cells derived from human induced pluripotent stem cells as a long-term host. Virol J. 2016;13:59.
107. Drukker M. Immunogenicity of embryonic stem cells and their progeny. Methods Enzymol. 2006;420:391–409.
108. Rong Z, Wang M, Hu Z, et al. An effective approach to prevent immune rejection of human ESC-derived allografts. Cell Stem Cell. 2014;14:121–130.
109. Fu X. The immunogenicity of cells derived from induced pluripotent stem cells. Cell Mol Immunol. 2014;11:14–16.
110. Jin X, Lin T, Xu Y. Stem cell therapy and immunological rejection in animal models. Curr Mol Pharmacol. 2015.
111. Dambacher S, de Almeida GP, Schotta G. Dynamic changes of the epigenetic landscape during cellular differentiation. Epigenomics. 2013;5:701–713.
112. de Almeida PE, Ransohoff JD, Nahid A, et al. Immunogenicity of pluripotent stem cells and their derivatives. Circ Res. 2013;112:549–561.
113. Zhang G, Shang B, Yang P, et al. Induced pluripotent stem cell consensus genes: implication for the risk of tumorigenesis and cancers in induced pluripotent stem cell therapy. Stem Cells Dev. 2012;21:955–964.
114. Tan Y, Ooi S, Wang L. Immunogenicity and tumorigenicity of pluripotent stem cells and their derivatives: genetic and epigenetic perspectives. Curr Stem Cell Res Ther. 2014;9:63–72.
115. Li L, Baroja ML, Majumdar A, et al. Human embryonic stem cells possess immune-privileged properties. Stem Cells. 2004;22:448–456.
116. Wu DC, Boyd AS, Wood KJ. Embryonic stem cells and their differentiated derivatives have a fragile immune privilege but still represent novel targets of immune attack. Stem Cells. 2008;26:1939–1950.
117. Drukker M, Katz G, Urbach A, et al. Characterization of the expression of MHC proteins in human embryonic stem cells. Proc Natl Acad Sci U S A. 2002;99:9864–9869.
118. Suarez-Alvarez B, Rodriguez RM, Calvanese V, et al. Epigenetic mechanisms regulate MHC and antigen processing molecules in human embryonic and induced pluripotent stem cells. PLoS One. 2010;5:e10192.
119. Lee AS, Tang C, Rao MS, et al. Tumorigenicity as a clinical hurdle for pluripotent stem cell therapies. Nat Med. 2013;19:998–1004.
120. Cui L, Shi Y, Zhou X, et al. A set of microRNAs mediate direct conversion of human umbilical cord lining-derived mesenchymal stem cells into hepatocytes. Cell Death Dis. 2013;4:e918.
121. Doi D, Morizane A, Kikuchi T, et al. Prolonged maturation culture favors a reduction in the tumorigenicity and the dopaminergic function of human ESC-derived neural cells in a primate model of Parkinson's disease. Stem Cells. 2012;30:935–945.
122. Martin GR. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci U S A. 1981;78:7634–7638.
123. Ben-David U, Benvenisty N. The tumorigenicity of human embryonic and induced pluripotent stem cells. Nat Rev Cancer. 2011;11:268–277.
124. Chinzei R, Tanaka Y, Shimizu-Saito K, et al. Embryoid-body cells derived from a mouse embryonic stem cell line show differentiation into functional hepatocytes. Hepatology. 2002;36:22–29.
125. Amariglio N, Hirshberg A, Scheithauer BW, et al. Donor-derived brain tumor following neural stem cell transplantation in an ataxia telangiectasia patient. PLoS Med. 2009;6:e1000029.
126. Laurent LC, Ulitsky I, Slavin I, et al. Dynamic changes in the copy number of pluripotency and cell proliferation genes in human ESCs and iPSCs during reprogramming and time in culture. Cell Stem Cell. 2011;8:106–118.
Copyright © 2016 Wolters Kluwer Health, Inc. All rights reserved.