Cell-based liver therapies: past, present and future

Abstract

Liver transplantation represents the standard treatment for people with an end-stage liver disease and some liver-based metabolic disorders; however, shortage of liver donor tissues limits its availability. Furthermore, whole liver replacement eliminates the possibility of using native liver as a possible target for future gene therapy in case of liver-based metabolic defects. Cell therapy has emerged as a potential alternative, as cells can provide the hepatic functions and engraft in the liver parenchyma. Various options have been proposed, including human or other species hepatocytes, hepatocyte-like cells derived from stem cells or more futuristic alternatives, such as combination therapies with different cell types, organoids and cell–biomaterial combinations. In this review, we aim to give an overview of the cell therapies developed so far, highlighting preclinical and/or clinical achievements as well as the limitations that need to be overcome to make them fully effective and safe for clinical applications.

This article is part of the theme issue ‘Designer human tissue: coming to a lab near you’.

1. Introduction

Orthotopic liver transplantation (OLT) represents the treatment of choice for patients with end-stage liver disease and several liver-based metabolic disorders. However, there is a significant scarcity of liver donors, which limits the number of patients who can benefit from this technique. Furthermore, OLT has additional disadvantages, such as the high cost of the procedure, the risks of complications related to surgery, and the need for lifelong immunosuppression, negatively affecting the patient's quality of life. Auxiliary liver transplantation, by just using a partial liver lobe and leaving the native liver in place in patients with acute liver failure and liver-based metabolic defects, is evidence that whole liver replacement is not necessary for the restoration of liver function. The partial lobe that is transplanted can provide the recipient with adequate liver function even in the cases of patients with gene defects. As such, this raises the option of using hepatocytes as a source of cells for the treatment of liver disease. Alternatives to OLT have long been desired and cell therapy has been suggested as one of the most promising approaches, being less invasive and potentially equally curative to liver transplantation. The first form of cell therapy for a liver disease that has been investigated was done using human primary hepatocyte transplantation (HTx). This approach was tested in a multitude of liver diseases both in preclinical and clinical settings, with promising results. However, it had shown some of the limitations described for OLT. Firstly, the shortage of liver tissues from which hepatocytes of good quality can be isolated represents an issue, as the clinical outcome of HTx strictly depends on the quality of cells transplanted. In addition, rejection against allogeneic cell graft is still limiting the long-term efficacy of this approach. As a consequence, other cell types have been considered, mostly focusing on the therapeutic potential of stem cells. They offer many advantages, being readily available and having the capacity to expand in vitro and in vivo. Furthermore, some of them can be isolated from the recipient thus allowing an autologous cell transplantation that would eliminate the need for immunosuppression. Stem cells under investigation for regenerative medicine applications include embryonic stem cells (ESC), bone marrow-derived haemopoietic cells (HSC), mesenchymal stromal cells (MSC) and, most recently, induced pluripotent stem cells (iPSC), derived from mature differentiated human cells re-programmed into an embryonic state and then differentiated into hepatocyte-like cells (HLC). Exciting results have been obtained in preclinical and, in some cases, clinical studies using stem cells. However, some limitations to their translation still exist, mainly related to the tumorigeneic potential and, in certain cases, their immature phenotype.

The aim of this review is to summarize the state of the art of cell therapies developed for the treatment of liver disease, highlighting their advantages and disadvantages and how, potentially, some of their limitations may be overcome. Furthermore, we have included a session describing new potential therapies that may become a reality in the future, including combination therapies, organoids or biomaterial-combined therapies.

2. Hepatocyte transplantation

HTx has emerged as a potential alternative to liver transplantation, as hepatocytes represent the major functional component of the liver and can provide the missing hepatic functions once engrafted. Hepatocytes can be isolated from donor livers rejected/unsuitable for OLT, using a three-step collagenase perfusion technique [1]. Once released, liver cells are filtered and centrifuged at low speed to separate hepatocytes from non-parenchymal cells. Hepatocytes can be used fresh for transplantation or cryopreserved for future use. HTx offers many advantages compared to OLT. Firstly, it is less invasive and less expensive, as it does not require complex surgery. Secondly, native liver remains in place allowing potential regeneration in patients with acute liver failure or serving as a backup in case of cell graft failure. Furthermore, it could represent a potential target for future gene therapy. In addition, HTx can be performed repeatedly if required and has the great advantage of being highly available, as the cells can be cryopreserved and used in case of emergency. It has been estimated that the level of cell replacement required to achieve physiological benefits is around 5–10% of the liver mass, thus multiple patients can be treated from one donor liver, saving other organs for patients who strictly need OLT.

The first preclinical study of HTx was performed in 1977 [2] in an animal model of Crigler–Najjar syndrome, a metabolic disorder due to a defective UDP-glucuronosyltransferase enzyme, determining increased unconjugated bilirubin levels and risk of death. The investigators observed a 35% decrease in bilirubin levels 28 days after hepatocytes transplantation. This and other preclinical investigations [3–5] led to the first human HTx in humans in 1992, when 10 patients with liver cirrhosis were transplanted with autologous hepatocytes, with unclear clinical benefits [6]. This was the first of a long list of human HTx in patients with liver disease, with more than 100 cases treated worldwide so far both for liver-based metabolic diseases and acute liver failure (table 1) [30]. The most promising results were shown when HTx was used for the treatment of metabolic disorders, in which the number of hepatocytes needed to counterweigh for a single-gene defect is lower than in the case of acute liver failure. Indications for HTx included urea cycle disorders [23–25], glycogen storage disease type 1 [17], factor VII deficiency [16], infantile Refsum's disease [19], phenylketonuria [21], severe infantile oxalosis [20], as well as acute liver failure [7–10]. Cells were most frequently transplanted by intraportal infusion, however, other routes—e.g. intraperitoneal [8,10] or intrasplenic [7,11] administration—were used when a higher number of cells was required or when coagulopathy issues or altered liver architecture may negatively impact on cell engraftment, as in acute liver failure or liver cirrhosis. Most centres use a dose up to 2 × 10⁸ cells kg⁻¹ of body weight as the recommended safe limit for HTx, however, multiple infusions can be performed when a high number of cells is required to ensure clinical benefits.

Table 1.

Liver disease treated using hepatocyte transplantation.

disease

— acute liver failure (drug [7], viral [7], idiopathic [8], mushroom poisoning [9], acute fatty liver of pregnancy [10]) — alpha1-antitrypsin [11] — Crigler-Najjar syndrome type I [12–14] — familial hypercholesterolemia [15] — factor VII deficiency [16] — glycogen storage diseases [17,18] — infantile Refsum's disease [19] — primary oxalosis [20] — phenylketonuria [21,22] — progressive familial intrahepatic cholestasis (A Dhawan 2013, unpublished data) — urea cycle defects [22–29]

Even though the safety of clinical human HTx was very well established, and some clinical benefits were appreciated, the improvement of hepatic functions was lost in the long term, usually within 1 year after cell transplantation, most probably due to poor cell engraftment and rejection against cell grafts, despite immunosuppression [31].

Cell engraftment can be highly influenced by the quality of hepatocytes, which tends to be quite poor when cells are isolated from liver tissues rejected for OLT, mostly because of prolonged ischaemia time, severe fatty liver, non-heart beating or elder donors. Therefore, the evaluation of cell quality before HTx is crucial. Many centres use trypan blue to assess cell viability, usually accepting cell batches with values equal or higher than 60% for clinical uses. However, other assays have been developed to better characterize the cells before HTx, including early signs of apoptosis and metabolic functions [32].

Cell rejection is another common cause of failure of HTx. Unlike the liver, which is an immune-privileged organ allowing in some cases graft tolerance and withdrawal of immunosuppression, allogenic hepatocytes are highly immunogenic [33]. Rejection against hepatocytes seems to be played by both the innate and adaptive immunity. Several mechanisms are involved, including an early cytolysis mediated by monocytes and granulocytes activated by surface adhesion proteins expressed on hepatocyte cell membrane [34], as well as the ‘instant blood-mediated inflammatory reaction’, in which the coagulation and complement systems are activated and result in the loss of up to 70% of transplanted cells [35,36]. In addition, allogeneic hepatocytes are also rejected by the adaptive immune system, most probably due to T-cell-mediated responses [26,37]. So far, immunosuppressive protocols used for OLT have been adapted for HTx and mostly consisted in the use of steroids and calcineurin inhibitors [12,16,17,24]. However, a better understanding of the immunological mechanisms involved in cell rejection and improvement of immunosuppressive regimens would probably improve HTx outcome.

A high grade of cell engraftment is another important prerequisite for HTx success. Several preconditioning regimens have been tried in order to provide a selective advantage to the transplanted cells over the recipient's ones, such as portal embolization [38], partial hepatectomy [39] and liver irradiation [22]. Portal vein embolization (PVE) is a safe and well-tolerated procedure commonly performed in patients undertaking liver resection, when the residual liver is not adequate in size to provide all the hepatic functions [40]. PVE determines hypertrophy of non-embolized liver segments thus giving a selective advantage to the cells transplanted in those lobes. This was shown by Dagher et al. [38] in Macaca monkeys, where the PVE of 50% of the liver followed by HTx resulted in 10% of cell replacement in the non-embolized lobe. Partial hepatectomy has been recently used in two patients with Crigler–Najjar syndrome type I before HTx. Although immunosuppression was discontinued in one patient leading to cell graft failure, the second patient showed a significant decrease in bilirubin levels for more than six months. However, he underwent OLT around 2 years later, as cell graft functions were undetectable [39]. Finally, irradiation of the native liver has emerged as a promising preconditioning approach to increase the engraftment of allogeneic hepatocytes after HTx. Recently, three patients were pre-treated with liver irradiation before HTx. Two of them—two infants with urea cycle defects—showed early graft loss, probably due to cell rejection, as shown by the increase of CD154⁺ T-cytotoxic memory cells (TcM). However, the third patient—an adult with phenylketonuria—preconditioned with liver irradiation before HTx and receiving more frequent immunosuppression adjustments, showed a decrease of phenylalanine levels in the blood for more than a year and clusters of proliferating transplanted hepatocytes in the liver. Nevertheless, cell graft was lost when follow-up became inconsistent [22].

Recently, alternative sources of hepatocytes have been explored to overcome the limitations due to the limited supply of human tissues and the frequently poor quality of cells isolated from adult livers. Fetal and neonatal livers have been considered as potential cell sources [41–43], because they are not currently considered for OLT. Although hepatic functions are not fully mature in these cells, neonatal hepatocytes have shown some hepatic detoxifying functions [41]. Hepatocyte xenotransplantation has recently been proposed as an alternative to human cells for HTx. Xenotransplantation would overcome the limitations related to human organ shortage, however, hepatocytes of other species—e.g. pigs—show some differences compared with human cells in terms of physiological activity, such as different coagulation system as well as metabolic and synthetic ability, and immunological constraints [44,45]. Furthermore, safety issues still exist, as there is the risk of transmission of infectious agents between species (xenozoonosis), as for instance porcine endogenous retroviruses. Hepatocyte xenotransplantation has never been tested in humans so far. However, it may become available once these limitations are overcome.

More recently, liver-humanized animal chimaeras have been developed, based on the knockout of the fumarylacetoacetate hydrolase (Fah) gene, which leads to a defect in the tyrosine catabolic pathway. These animals show an extensive level of liver repopulation when transplanted with wild-type hepatocytes [46]. Furthermore, they can be repopulated with human hepatocytes when animals are genetically modified with immune-deficiency alleles (e.g. chimaeric FRG mouse) [47]. These animal models could be used as living bioreactors to produce large quantities of human hepatocytes that are fully mature and functional. However, it is important to mention that some level of animal cell contamination will be present as animal's liver repopulation is never fully complete, therefore the same safety issues mentioned for xenotransplantation must be considered, even though to a lesser extent.

3. Haematopoietic and mesenchymal stem cells as cell sources for liver cell therapy

Haematopoiesis is the process by which blood cells develop. It is a complex process involving self-renewal, differentiation and maturation [48]. Haematopoiesis is governed by many cytokines that promote the survival, proliferation and differentiation of haematopoietic stem and progenitor cells. Haematopoietic stem cells (HSC) are defined as cells that can generate differentiated progeny of multiple blood cell lineages (lymphoid and myeloid) as well as the potential to self-renew. The capacity to self-renew enables the HSC population to sustain haematopoiesis throughout life [49,50].

In the human embryo, erythropoietic cells first appear within the blood islands of the yolk sac about 19 days after fertilization. Haematopoietic foci develop in the hepatic cord during the sixth week of gestation, and the liver becomes the major site of erythropoiesis in the middle trimester of pregnancy. During this period, about half the nucleated cells of the liver consist of erythroid cells. A few myeloid cells and megakaryocytes are also found. The number of erythroid cells in the liver decreases progressively after the seventh month of gestation. After the sixth month, the bone marrow becomes the major site of haematopoiesis [51].

Given the origins of haematopoiesis, embryonically within the liver, HSCs have been used clinically for the treatment of liver disease. There are two main rationales for using these HSC in liver regeneration. The first, HSC are known to participate in liver regeneration [52–54]. The second being evidence from animal models of liver injury, in which HSC infusion can rescue injury and improve outcomes [55–57].

The identification and purification of HSC have been an active field of research over the last two decades. At present, the precise phenotype of the HSC is incompletely defined. The description of a haematopoietic stem cell has essentially reflected methods, techniques and hardware available to investigators [58]. Various assays have been developed to characterize HSC. As a result, the ambiguous use of the term HSC coupled with the infusion of other stem types for liver function restoration makes interpretation of outcomes more complex [59]. In spite of these ambiguities, Cluster of Differentiation (CD) markers on cells are used as surrogate HSC markers. These include CD34 and CD133 as markers of HSC and used in cell infusion for liver disease [60–62]. Recently, it has been shown that transplantation of CD133 bone marrow-derived cells can transiently improve end-stage liver disease [63]. Granulocyte-Colony Stimulating Factor autologous mobilized peripheral blood and CD34+ cells have also been used to treat liver disease with some success [64]. At present, there are 17 clinical trials registered on the Clinicaltrials.gov website for the use of HSC in the treatment of various liver diseases (table 2). It has been shown that derivatives of HSC such as macrophages can improve outcomes of liver fibrosis in mice [67]. The exact mechanism of the effect of transplanted and differentiated macrophages and host hepatocytes remain to be elucidated. It is suggested that a paracrine mode of action of HSC on resident cells enables liver regeneration [68]. A further caveat to HSC transplantation for liver disease is the need for immunosuppression, although this may be circumvented through the use of induced pluripotent HSC.

Table 2.

Clinical trials on Clinicaltrials.gov completed or recruiting for the treatment of liver disease with bone marrow stem cells or mesenchymal stromal cells.

NCT number	title	recruitment	conditions	interventions	age	phases	enrolment	start date	locations	outcome
NCT00147043	adult stem cell therapy in liver insufficiency	completed	liver cirrhosis	infusion of CD34+ stem cells via image-guided scan	20 years to 65 years (adult)	phase 1	5	Jan 2005	Hammersmith Hospitals NHS Trust, London, United Kingdom	not available on Clinicaltrials.gov
NCT00655707	a phase I/II safety and tolerability dose escalation study of autologous stem cells to patients with liver insufficiency	completed	liver disease	autologous expanded CD34+ haemopoietic cells transplantation	20 years to 65 years (adult)	phase 1|phase 2	5	Jan 2006	Imperial College Healthcare Trust, London, United Kingdom	not available on Clinicaltrials.gov
NCT00713934	autologous bone marrow stem cells in cirrhosis patients	completed	liver cirrhosis	CD133+ stem cells	20 years to 65 years (adult)	phase 1|phase 2	7	Jan 2008	Liver Transplant Research Center, Shiraz, Fars, Iran|Royan Institute, Tehran, Iran	no adverse effects; no significant alterations of liver function parameters [65]
NCT01120925	autologous bone marrow-derived stem cells in decompensate cirrhotic patients	completed	liver cirrhosis	CD133+ stem cells transplantation	16 years to 65 years (child, adult)	phase 1|phase 2	30	May 2010	Gastroenterology and Hepatic Disease Research Center, Tehran, Iran	not available on Clinicaltrials.gov
NCT01745731	cell infusion intraportal autologous bone marrow mononuclear as enhancer of liver regeneration	completed	liver transplant rejection	cell infusion intraportal mononuclear bone marrow autologous and portal embolization of the affected segments	18 years and older (adult, senior)	phase 2	13	Mar 2011	University Hospital Virgen del Rocio, Sevilla, Spain	not available on Clinicaltrials.gov
NCT02943707	autologous bone marrow stem cells infusion for the treatment of liver diseases	recruiting	liver diseases	autologous bone marrow stem cells infusion	18 years to 60 years (adult)	phase 2	40	Oct 2016	The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China	autologous MSC transplantation is safe for liver failure patients caused by chronic hepatitis B; short-term efficacy was favourable, but long-term outcomes were not markedly improved [65]
NCT02260375	MSC therapy in liver transplantation	recruiting	liver transplant rejection	mesenchymal stromal cells	18 years and older (adult, senior)	phase 1	20	Oct 2014	Ospedale di Bergamo, Italy|Ospedale di Bologna, Italy	not available on Clinicaltrials.gov
NCT03209986	trial of mesenchymal stem cell transplantation in decompensated liver cirrhosis	recruiting	liver cirrhosis	mesenchymal stem cell transplantation via peripheral vein	18 years to 65 years (adult)	phase 1	200	Aug 2017	Xijing Hospital of Digestive Disease, Xijing Hospital, Xi'an, Shaanxi, China	not available on Clinicaltrials.gov
NCT02705742	mesenchymal stem cells transplantation for liver cirrhosis owing to HCV hepatitis	recruiting	liver cirrhosis	mesenchymal stromal cells	18 years to 70 years (adult, senior)	phase 1|phase 2	5	Jan 2016	Gulhane Military Medical Academy, Ankara, Turkey	no side effect observed; in 8 patients out of 12, improvements in model for end-stage liver disease scores were observed [66].
NCT01342250	human umbilical cord mesenchymal stem cells transplantation for patients with decompensated liver cirrhosis	completed	liver cirrhosis	conventional therapy plus hUC-MSCs treatment	18 years to 70 years (adult, senior)	phase 1|phase 2	20	Oct 2010	Shanghai Liver Disease Research Center, Shanghai, China	not available on Clinicaltrials.gov
NCT03254758	a study of ADR-001 in patients with liver cirrhosis	recruiting	decompensated liver cirrhosis	mesenchymal stromal cells	20 years and older (adult, senior)	phase 1|phase 2	15	July 2017	Niigata University Medical & Dental Hospital, Niigata, Japan	not available on Clinicaltrials.gov
NCT00420134	improvement of liver function in liver cirrhosis patients after autologous mesenchymal stem cell injection: a phase I–II clinical trial	completed	liver failure|cirrhosis	injection of progenitor of hepatocyte derived from mesenchymal stem cell	18 years and older (adult, senior)	phase 1|phase 2	30	Feb 2006	Research Center for Gastroenterology and Liver Diseases, Tehran, Iran	not available on Clinicaltrials.gov
NCT01844063	safety and efficacy of diverse mesenchymal stem cells transplantation for liver failure	recruiting	liver failure	conventional plus BM-MSC treatment	18 years to 65 years (adult)	phase 1|phase 2	210	Jul 2013	Qi Zhang, Guangzhou, Guangdong, China	not available on Clinicaltrials.gov
NCT01591200	dose finding study to assess safety and efficacy of stem cells in liver cirrhosis	completed	alcoholic liver cirrhosis	allogeneic mesenchymal stem cells	18 years to 65 years (adult)	phase 2	40	Jun 2012	India	not available on Clinicaltrials.gov
NCT01875081	REVIVE (Randomized exploratory clinical trial to evaluate the safety and effectiveness of stem cell product in alcoholic liver cirrhosis patient)	completed	alcoholic liver cirrhosis	livercellgram (MSC)	20 years to 70 years (adult, senior)	phase 2	72	Nov 2012	Pharmicell Co. Ltd., Seoul, Korea	not available on Clinicaltrials.gov
NCT02957552	safety and tolerance of immunomodulating therapy with donor-specific MSC in pediatric living-donor liver transplantation	recruiting	pediatric liver transplantation	mesenchymal stem cells	8 weeks to 18 years (child, adult)	phase 1	7	Mar 2017	University children's Hospital, Tuebingen, Germany	not available on Clinicaltrials.gov
NCT00956891	therapeutic effects of liver failure patients caused by chronic hepatitis B after autologous MSCs transplantation	completed	liver failure	mesenchymal stem cells	15 years to 70 years (child, adult, senior)	phase 1|phase 2	158	May 2005	The Third Affiliated Hospital Of Sun Yat-sen University, Guangzhou, Guangdong, China	not available on Clinicaltrials.gov
NCT02857010	allogenic bone marrow mesenchymal stem cell therapy in acute-on-chronic liver failure	recruiting	acute-on-chronic hepatic failure	allogenic mesenchymal stem cells	18 years to 79 years (adult, senior)	phase 1	30	Feb 2016	Hospital Clinic de Barcelona, Barcelona, Spain	not available on Clinicaltrials.gov
NCT01454336	transplantation of autologous mesenchymal stem cells in decompensate cirrhotic patients with pioglitazone	completed	liver fibrosis	mesenchymal stem cells	18 years to 65 years (adult)	phase 1	3	Jun 2010	Royan Institute, Tehran, Iran	not available on Clinicaltrials.gov
NCT02706132	therapeutic strategy and the role of mesenchymal stromal cells for ABO incompatible liver transplantation	recruiting	liver transplantation	mesenchymal stem cells (MSCs)	18 years to 60 years (adult)	phase 1|phase 2	15	Feb 2014	The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China	not available on Clinicaltrials.gov
NCT02806011	long-term follow-up study of livercellgram in alcoholic liver cirrhosis patients who completed livercellgram phase 2 study	enrolling by invitation	alcoholic liver cirrhosis	livercellgram (MSC)	20 years to 70 years (adult, senior)	phase 2	50	May 2016	not provided	not available on Clinicaltrials.gov
NCT01062750	liver regeneration therapy by intrahepatic arterial administration of autologous adipose tissue-derived stromal cells	completed	liver cirrhosis	adipose tissue-derived stromal cells dosage	20 years to 80 years (adult, senior)	phase 1	4	Oct 2012	Kanazawa University Hospital, Kanazawa, Ishikawa, Japan	not available on Clinicaltrials.gov
NCT02223897	mesenchymal stem cells transplantation for ischaemic-type biliary lesions	recruiting	ischaemic-type biliary lesions	HUC-MSCs	18 years to 60 years (adult)	phase 2|phase 3	66	July 2014	The Third Affiliated Hospital, Sun Yat-Sen University, Guangzhou, Guangdong, China	not available on Clinicaltrials.gov
NCT02652351	human umbilical cord-mesenchymal stem cells for hepatic cirrhosis	recruiting	hepatic cirrhosis	human umbilical cord mesenchymal stem cells	18 years to 80 years (adult, senior)	phase 1	20	Mar 2016	The Second Affiliated Hospital of University of South China, Hengyang, Hunan, China	not available on Clinicaltrials.gov
NCT02786017	injectable collagen scaffold combined with HUC-MSCs transplantation for patients with decompensated cirrhosis	recruiting	decompensated cirrhosis	injectable collagen scaffold+HUC-MSCs	18 years and older (adult, senior)	phase 1|phase 2	40	May 2016	The Affiliated Nanjing Drum Tower Hospital of Nanjing, Jiangsu, China	not available on Clinicaltrials.gov
NCT01378182	efficacy of in vitro expanded bone marrow-derived allogeneic mesenchymal stem cell transplantation via portal vein or hepatic artery or peripheral vein in patients with Wilson cirrhosis	completed	Wilson's disease	allogeneic mesenchymal stem cell transplantation	18 years to 65 years (adult)	phase 2	10	Apr 2011	Department of Gastroenterology; Gulhane Military Medical Academy, Ankara, Turkey	not available on Clinicaltrials.gov

Mesenchymal stem cells, also referred to as mesenchymal stromal cells or multipotent stromal cells (MSC), are a population of immature cells [69] that reside in many tissues, including adult bone marrow [70], adipose tissue [71], cartilage [72], umbilical cord, fetal liver and bone marrow [73] and adult liver. The common feature of MSC is the ability to differentiate into adipocyte, cartilage and osteogenic tissue. Culture protocols for MSC usually include direct plastic adherence or enrichment by CD marker selection or a combination thereof. These cultured MSC are quite heterogeneous by morphological analysis, mainly exhibiting fibroblastic shapes.

Significant research in rodent models and in humans has shown that these MSC are very well characterized by cell surface markers and function (clinical utilities). The frequency of MSC isolated from different sources of tissue, however, varies considerably from 0.01 to 0.0001% of nucleated cells in the adult bone marrow to 0.0005% of nucleated cells in first trimester fetal liver [74]. There is also considerable heterogeneity in MSC differentiation potential, in spite of similar phenotypic and antigenic profiles.

As MSC also are invariably immunologically naive, as reflected by the absence of HLA type II markers, substantial research has been done into the immunomodulatory effect of MSC in many diseases. In liver disease, it is suggested that as in HSC, a paracrine mode of action of MSC and donor tissue is responsible for liver regeneration. Considerable debate exists on the exact functional properties of MSC derived from the different sources of tissue and its role in liver regeneration.

As MSC have unique properties of being immunomodulatory particularly to sites of injury, it is an important resource for use in liver regeneration. Substantial clinical trials have been conducted and are currently being carried out to assess the properties of MSC in the treatment of liver disease (table 2).

4. Pluripotent stem cells: embryonic and induced cells as cell sources for liver regeneration

Pluripotent stem cells by definition are able to form cell types from all three germ cell layers, i.e. the endoderm, mesoderm and ectoderm [75]. During embryogenesis, the inner cell mass of the developing embryo is destined to become all of these cell types. As such, cells derived from the inner cell mass of a developing embryo and in vitro cultured result in the creation of ESC. These cells in culture and in vivo have the ability to form all three major germ cell components of the body. The regenerative properties of these ESC cells have been significant notwithstanding ethical issues.

An important aspect of ESC research is the ability to differentiate into a particular cell type of interest. The differentiation of ESC into hepatocytes has been very well studied in vitro. In vivo studies have shown the promise of these cells in the treatment of liver disease. However, translation of ESC research into the clinic is somewhat limited, particularly in the field of liver disease.

Just over a decade ago, Yamanaka and colleagues were able to show that mature differentiated cell types could be re-programmed into an embryonic state. This was done through the delivery of four transcription factors into mature cells that resulted in an embryonic phenotype. These cells were termed induced pluripotent cells (iPSC), as they were able to show that these iPSC could then differentiate into all three germ layers [76–78]. As a result of this major breakthrough and paradigm shift of the plasticity of human cells, much progress has been made by virtue of developing iPSC towards the clinic. In the field of liver regeneration, iPSC can be readily differentiated into HLC [79,80] and not only on a large scale but using processes that are clinically accepted [80]. There are planned and limited clinical trials on HLC for end-stage liver disease. Most of the research is now focusing on the improvement of their metabolic functions, in order to make them as effective as primary human hepatocytes and to limit their tumorigenic potential, which still represents a risk.

More recently, it has been revealed that bacteria and archaea have an adaptive immune system that can recognize a previous infection from a bacteriophage. The mechanism of this process has been elucidated by the virtue that bacteria are able to edit genomes of invading bacteriophages. This is done using a relatively simple way of snipping the DNA of an invading bacteriophage and inserting the snipped sequences within the bacterial genome itself. These sets of sequences are termed clustered, regularly interspaced, short palindromic repeats (CRISPR). A subsequent challenge of the same pathogen to the bacteria will result in the editing and destruction of the invading pathogen using a Cas-9 enzyme system encoded within the bacteria genome as well. This system allows bacteria to have memory of invading pathogens [81,82].

From this incredible discovery, scientists have then exploited this ability to edit various genomes for the purpose of genetic modification. Much progress has been made into using gene knock-in and knock-out techniques to correct genetic defects. The CRISPR/Cas-9 system combined with a guide RNA targeting a particular genetic locus can be efficiently used to correct genetic defects found in rodents and humans [83,84].

Given the ability to efficiently genetically modify the genome through the CRISPR/Cas-9 system, many genetic disorders in humans could easily be corrected. Therefore, it may be possible to obtain patient-specific iPSC and specifically correct the defects via the CRISPR/Cas-9 system. The corrected patient iPSC could then be differentiated into the appropriate cell type for re-infusion into patients, thereby correcting the disorder. This approach has been used to correct a 3 base-pair deletion in the gene coding for low-density lipoprotein receptor (LDLR) in iPSC derived from a patient with familial hypercholesterolemia. In vitro, HLC derived from corrected iPSC restored LDLR-mediated endocytosis [85]. Similarly, the CRISPR/Cas-9 system has been used to correct iPSC derived from three patients with arginase deficiency, an autosomal recessive disorder affecting the final step of the urea cycle. The authors showed a universally applicable strategy to correct the disease, based on the addition of a full-length codon-optimized human arginase 1 cDNA into the exon 1 of the gene coding for hypoxanthine-guanine phosphoribosyltransferase (HPRT) through the use of Cas-9 nickases, able to bind and cleave HPRT in a targeted way. Once corrected, both iPSC and differentiated HLC demonstrated the restoration of arginase activity [86]. The challenge now is to bring such incredible scientific breakthroughs to the clinic. Considerable progress has been made in this area and several clinical trials are expected in the next decade.

5. Future developments

In the last two decades, cell-based therapies for the treatment of liver disease drew considerable interest as a potentially effective alternative to OLT. However, the therapies developed so far still have limitations, such as (i) poor cell engraftment and long-term efficacy in case of allogenic HTx, (ii) safety issues for animal-derived hepatocytes or (iii) potential tumorigenesis for stem cell-derived HLC. Several alternative approaches are being considered to widen the options available and potentially improve the efficacy of cell-based therapies for liver diseases. Combination therapies, organoids and biomaterial-combined cell therapies represent some of these options currently under investigation, and may be translated into clinical applications in the near future.

MSC have already been used in clinical settings for the treatment of liver disease, due to their enormous immunomodulatory and anti-inflammatory potential, as described above. However, quite recently MSC have also been proposed as a way to induce tolerance in organ transplantation (e.g. kidney and liver) with promising results [87]. In addition, MSC have been shown to prevent rejection of allogeneic islet grafts in vivo and to improve transplanted islet functions [88,89]. A similar approach consisting of the combination of allogeneic hepatocytes with MSC for cell transplantation may be considered in the future, with the aim of decreasing cell rejection. Furthermore, MSC have been shown to exert a significant improvement on hepatocyte functions both in vitro and in vivo; therefore, combining them with hepatocytes could further improve HTx outcome [90].

An additional advance in the field of regenerative medicine has been the ability to grow miniature organ-like tissues in the laboratory. In 2013, Takebe and colleagues were able to show that liver buds with a vasculature system could be grown in a dish using hepatocytes derived from iPSC, human endothelial vasculature cells and MSC in Matrigel [91]. These liver buds were termed organoids and could be transplanted in a drug-induced mouse liver failure model, thereby rescuing liver failure. Significant research in this field has been made including using the intestine and lung [92,93]. However, challenges still remain in translating these findings into the clinic, such as the ability to culture these organoids in a clinically accepted manner and to find a suitable route of delivery.

Biomaterial-combined cell therapies are currently of high interest for regenerative medicine. A wide range of biomaterials can be used for different purposes. Alginate, a natural polysaccharide derived from brown seaweed, is being investigated as a biocompatible hydrogel to encapsulate human or other species hepatocytes in small microbeads for intraperitoneal transplantation in patients with acute liver failure [94,95]. This approach would allow donor cells to perform all the hepatic functions while the recipient's liver regenerates. At the same time, alginate would protect transplanted cells from the host immune cells, thus avoiding the need for immunosuppression which may be detrimental in very sick patients with acute liver failure. This approach was effective in a rat model of acute liver injury [94,96]. In addition, the possibility of cryopreserving microbeads containing hepatocytes with the maintenance of cell function after microbead thawing has been recently described [97]. Another interesting biomaterial under investigation is decellularized livers. These tissues could be used as a scaffold and repopulated with cells before transplantation, with the assumption that providing a three-dimensional non-immunogenic structure rich in extracellular matrix proteins to allogeneic cells would improve their engraftment and functions [98].

6. Conclusion

There has been a considerable development of cell therapies for the treatment of liver disease in the last two decades. The first and most established option used both in preclinical and clinical settings is human HTx, which has shown short-term clinical and overall safety improvements. However, it has failed so far in terms of long-term efficacy. New approaches to improve hepatocyte quality and their engraftment are needed to prolong HTx clinical benefits.

Other cell types, mainly stem cell-derived, have been proposed as an alternative to hepatocytes for cell transplantation. They are very attractive to the scientific community worldwide because of their high availability, good cell quality, as well as the possibility to use them in autologous cell transplantation. However, various hurdles need to be overcome to fully translate their use into routine clinical practice, mainly related to limited efficacy and/or inadequate safety.

Novel approaches such as combinations of hepatocytes with other cell types or with biomaterials are being investigated and may become a valid alternative to the options currently available. This may improve the efficacy of cell transplantation for liver disease and widen its clinical applications.

Data accessibility

This article has no additional data.

Authors' contributions

V.I. and A.C. contributed equally to drafting and writing the review; A.D. critically revised and approved the final version for publication.

Competing interests

The authors have no competing interests.

Funding

The authors acknowledge ‘MowatLabs’ for financial support.