Hepatocyte transplantation and advancements in alternative cell sources for liver-based regenerative medicine

Hepatocyte transplantation and advancements in alternative cell sources for liver-based... Human hepatocyte transplantation has been actively perused as an alternative to liver replacement for acute liver failure and liver-based metabolic defects. Current challenges in this field include a limited cell source, reduced cell viability following cryopreservation and poor engraftment of cells into the recipient liver with consequent limited life span. As a result, alternative stem cell sources such as pluripotent stem cells, fibroblasts, hepatic progenitor cells, amniotic epithelial cells and mesenchymal stem/stromal cells (MSCs) can be used to generate induced hepatocyte like cells (HLC) with each technique exhibiting advantages and disadvantages. HLCs may have comparable function to primary human hepatocytes and could offer patient-specific treatment. However, long-term functionality of transplanted HLCs and the potential oncogenic risks of using stem cells have yet to be established. The immunomodulatory effects of MSCs are promising, and multiple clinical trials are investigating their effect in cirrhosis and acute liver failure. Here, we review the current status of hepatocyte transplantation, alternative cell sources to primary human hepatocytes and their potential in liver regeneration. We also describe recent clinical trials using hepatocytes derived from stem cells and their role in improving the phenotype of several liver diseases. . . . . Keywords Hepatocyte transplantation Induced pluripotent stem cells Fibroblast Mesenchymal stem/stromal cell Hepatic progenitor cells Liver regeneration Abbreviations fVII Factor VII ALF Acute liver failure FGF Fibroblast growth factor BMM Bone marrow-derived macrophages FHF Fulminant hepatic failure BMP4 Bone morphogenetic protein 4 hAECs Hepatic differentiation of amniotic CN-1 Crigler-Najjar syndrome type 1 epithelial cells CYP Cytochrome p450 HPCs Hepatic progenitor cells DEX Dexamethasone HNF1A Hepatocyte nuclear factor 1 EGF Epidermal growth factor homeobox alpha HT Hepatocyte transplantation HGF Hepatocyte-growth factor HLCs Hepatocyte-like cells Siddharth Sinha and Charlotte A Lee are joint first authors iPSCs Induced pluripotent stem cells PE Plasma exchange * Anil Dhawan Anil.dhawan@kcl.ac.uk EpCAM Primary human bile duct cells CLiPs Proliferative bipotent cells Dhawan Lab, Institute of Liver Studies, King’s College London at MSCs Mesenchymal stem cells King’s College Hospital NHS Foundation trust, London, UK MSC-CM MSC-conditioned medium Paediatric Liver GI and Nutrition Centre, King’s College London at OSM Oncostatin M King’s College Hospital NHS Foundation Trust, London, UK OTC Ornithine transcarbamylase deficiency OLT Orthotopic liver transplantation UC-MSC Umbilical cord–mesenchymal stem cell 470 J Mol Med (2018) 96:469–481 Introduction the primary long-term treatment is liver transplantation, the lack of an appropriately sized, blood matched donor organ The liver is responsible for a diverse range of functions within can lead to irreversible neurological disability. There are re- the body ranging from xenobiotic metabolism to plasma pro- ports of 8 patients that have received between 1.4 and 7.5 tein synthesis [1]. However, the liver can become susceptible billion hepatocytes, which showed up to a 50% reduction in to acute failure (ALF) due to viral infection or drug effect bilirubin and UGT activity and a decrease in the need for amongst other aetiologies [2]. Liver-based metabolic disor- phototherapy. The majority of these patients went onto receive ders that are caused by single-gene defects can result in a lack an OLT within a year as the effects of HT were not long of specific liver-based enzyme function which may result in lasting, with one patient avoiding OLT for 4 years [16–21]. damage to other organs most often irreversible neurodisability Several urea cycle defects have also been shown to benefit [3]. Orthotopic liver transplantation (OLT) is currently the from hepatocyte infusions. This includes ornithine only viable treatment for severe ALF and certain liver-based transcarbamylase deficiency (OTC), which is an X-linked ge- metabolic disorders [2, 4, 5]. Liver replacement is not futuris- netic condition affecting males that causes hyperammonemia tic for liver-based metabolic defects where gene therapy and can also lead to neurological implications including devel- would be ideal. Notably, a shortage in healthy donor livers opmental delay and learning disabilities. HT led to decreased has led to research into alternative treatment options [6]. ammonia levels and increased urea production in six of these Hepatocyte transplantation (HT), where liver cells may pro- patients, with four receiving an OLT within 7 months and two vide a potential alternative to OLT or act as a bridge until a reported mortalities [14, 22–25]. Other metabolic liver diseases suitable organ becomes available, has been demonstrated, shown to improve following HT include familial hypercholes- with over 100 cases published worldwide showing the safety terolemia where there was up to a 20% decrease in LDL in and preliminary efficacy of the technique [7, 8]. three patients, fVII deficiency where there was a 70% decrease This review will focus on the current status of HT, alternate in the need for recombinant fVII for 6 months and ASL defi- cell sources to primary human hepatocytes and alternate ciency where the was a decrease in ammonia production and methods of liver regeneration which can be used either inde- increased psychomotor abilities [26–28]. Nevertheless, the pendently or in combination with HT. long-term benefits of HT for the treatment of patients with liver-based metabolic disease have yet to be shown. HT can also be used in patients with ALF. Although liver The current status of hepatocyte transplantation is the primary treatment, paediatric patients transplantation may die whilst waiting for an appropriately size and blood- matched liver, with no other treatment being shown to im- prove survival [29]. Furthermore, existing listing criteria are HT is a less invasive alternative to OLT. Hepatocytes are iso- lated from donor livers that have been rejected for organ trans- not robust in terms of accurately predicting death and survival; plantation in view of prolonged warm or cold ischaemia times, hence, some patients receive a liver transplantation, even if mild steatosis or aberrant anatomy. Hepatocytes isolated from their liver may spontaneously recover, leading to unnecessary one donor liver can yield a high quantity of cells and be used surgeries with life-long immunosuppression. Thirty-seven pa- to treat multiple patients [9, 10]. The ability to cryopreserve tients with acute liver failure have received human hepato- these cells is one of the major advantages of HT, potentially cytes for both drug induced, viral and idiopathic ALF. Ten providing an off-the-shelf treatment and enabling cells of all of these patients underwent intraportal hepatocyte infusions, blood groups to be immediately available for emergency with two making a full recovery without the need for OLT and cases, which is particularly important for patients with ALF. three were successfully bridged to transplantation [15, 30–33]. Several routes of infusion for cell transplantation have been However, there is a high risk associated with placing described including intraportal, intrasplenic and intraperitone- intrahepatic invasive catheters in coagulopathic patients. As al. Depending on the age of the patient, infusion of hepato- an alternative, it is possible to encapsulate hepatocytes in al- cytes into the portal vein can be undertaken via the umbilical ginate microbeads made from purified alginate using an vein under fluoroscopy to avoid a patent ductus venous. In encapsulator [34]. The semi-permeable membrane within the older children, radiological or surgical catheters are required microbeads allows exchange of metabolites, maintaining syn- using radiological guidance [11–13]. thetic detoxification function, whilst also protecting against Hepatocyte transplantation can be used to treat patients immunocompetent cells by preventing the entry of antibodies with liver-based metabolic diseases and ALF [7, 14, 15]. [35, 36]. Hepatocyte-containing microbeads are transplanted Metabolic liver diseases treated using HT include Crigler- into the intraperitoneal cavity. The transplanted hepatocytes Najjar syndrome type 1 (CN-1), urea cycle defects and factor can assist the necessary liver functions, allowing the liver to VII (fVII) deficiency [16]. Crigler-Najjar syndrome has an recover. After full recovery, usually 1 month, the microbeads incidence of 1 in 1,000,000 births in the UK, and although can be removed using a laparoscopic peritoneal lavage. J Mol Med (2018) 96:469–481 471 Despite extensive clinical data, long-term clinical out- hepatoblast formation and hepatocyte-like differentiation, re- comes of HT have yet to be established with any type of spectively. Si-Tayeb et al. showed that iPSC-derived HLCs liver-based metabolic disease or ALF. There are still many displayed hepatic functionality, including glycogen accumu- limitations to the technique, and current research aims to over- lation, lipoprotein uptake and urea synthesis [47]. iPSCs are come this. Firstly, the cryopreservation process requires fur- advantageous over primary human hepatocytes as generation ther optimisation with the current protocol resulting in low from somatic cells of an individual will prevent activation of viability and function of hepatocytes post-thawing. the recipient’s immune response, avoiding the use of immu- Furthermore, transplanted donor hepatocytes undergo im- nosuppressive drugs [38, 45, 48, 49]. iPSCs can proliferate mune rejection with up to 70% of engrafted cells cleared with- indefinitely, forming a limitless pool of HLCs allowing pa- in the first 24 h post-transplantation [37]. Upon transplanta- tients to receive multiple infusions if necessary [48, 50]. tion, donor hepatocytes are recognised and activate the instant iPSC-derived HLCs can also be used as a model for several blood-mediated inflammatory reaction, during which both the metabolic liver diseases including familial hypercholesterol- complement and coagulation pathways are activated. Innate emia, α -antitrypsin deficiency and glycogen storage disease immune cells such as Kupffer cells, natural killer cells and type 1a. By generating cells with similar phenotypes to those monocytes rapidly clear transplanted donor hepatocytes [38, caused by inherited diseases, it is possible to study the mech- 39]. A major limitation of HT is the lack of good quality donor anisms of dysregulated cellular functions and identify ways to organs from which to isolate cells from. Neonatal livers have treat or reverse the condition [51]. Currently, the functional been investigated as a potential cell source as they are infre- ability of HLCs is not comparable to human primary hepato- quently used for OLT due to the high incidence of hepatic cytes. Yu et al. showed that HLCs co-express alpha-fetopro- artery thrombosis [40]. However, they may be an ideal cell tein and albumin, suggesting that they are not fully mature. source for HT, with one study demonstrating their mature Levels of albumin synthesis, urea production, cytochrome function of cytochrome P450 and ureagenesis enzymes, with p450 activity and mitochondrial function are also significantly increased attachment efficiency and viability compared to he- lower than human primary hepatocytes [50]. Furthermore, patocytes isolated from adult donor livers [41, 42]. Currently, there are genetic variations in donor cells that effect the dif- the number of neonatal donations is not sufficient to maintain ferentiation propensity of iPSCs which has been attributed to an active clinical HT programme [41, 43]. As a result, there is genetic variation of the donor, cell culture conditions and a requirement to investigate alternative cell sources to resolve iPSC generation protocols [52, 53]. Following the develop- the shortage of healthy donor organs and avoid recipient im- ment of new hepatic differentiation protocols, Kajiwara et al. mune rejection (Fig. 1 and Table 1). compared 28 iPSC lines derived from different somatic cells and showed that it was the origin of the donor cells that deter- mined the variation in hepatic differentiation and not the der- Alternative cell sources for hepatocyte ivation method [53].With albumin used as a marker to assess transplantation functionality, HLCs derived from human dermal fibroblast- iPSCs and peripheral blood cell-iPSCs showed minimal vari- Induced pluripotent stem cells generated ation in hepatic differentiation from the same donor; however, from somatic cells inter-donor hepatic variation was more prominent. This cre- ates a complication when using iPSCs for therapeutic use as Induced pluripotent stem cells (iPSCs) that can be differenti- non-identical cell lines cannot guarantee the same quality of ated into hepatocyte-like cells (HLCs) are being widely ex- HLC production [48, 53]. plored as an alternative to primary human hepatocytes. These In addition, the tumorigenic potential of these cells due to cells offer an excellent alternative source of hepatocytes and the presence of oncogenes such as c-Myc may raise a safety are advantageous over primary human hepatocytes because of concern regarding the clinical application of these cells. Chen their unlimited cell source and their ability to avoid the im- et al. reported no formation of teratomas or tumours in Gunn mune system [44]. Methods to produce iPSC-derived HLCs rats transplanted with HLCs, suggesting a loss of pluripotent are well established, with most protocols using a three- characteristics within these cells [54]. Although HLCs repre- dimensional matrix such as Matrigel® to establish the gener- sent an ideal cell source for HT with no risk of rejection, work ation of hepatocytes. However 2D matrices including collagen is still ongoing to advance their functional capacity and fully have also shown successful iPSC to HLC differentiation [45, validate the safety and efficacy of using these stem cells. 46]. An effective protocol for generating HLCs has been established via the use of specific growth factors such as Fibroblasts activin A and bone morphogenetic protein 4 (BMP4), which are crucial for initiating the first step of hepatic maturation. Human fibroblasts offer another potential source of HLCs for Hepatocyte-growth factor (HGF) and oncostatin stimulate HT. Fibroblasts are connective tissue cells found in all areas of 472 J Mol Med (2018) 96:469–481 Fig. 1 Potential alternative cell sources (induced pluripotent stem cells, HPC hepatic progenitor cells, hAEC human amniotic epithelial cells, fibroblasts, mesenchymal stem/stromal cells and hepatic progenitor cells) BMP bone morphogenetic protein, OSM oncostatin M, HGF hepatic which can be used to generate hepatocytes. Gene transfer is used to growth factor, HNF1A hepatocyte nuclear factor 1 homeobox alpha, convert somatic cells to iPSCs and fibroblasts to HLCs. All other trans- HNF4A hepatocyte nuclear factor 4 alpha, FGF fibroblast growth factor, formations occur under culture conditions. HLC induced hepatocyte, EGF epidermal growth factor, Dex dexamethasone, FBS foetal bovine iPSC induced pluripotent stem cells, MSC mesenchymal stem cells, serum the human body [55]. Huang et al. established the first fibroblast reprogramming [56]. Simeonov et al. also achieved reprogramming of human fibroblasts to hepatocytes using fibroblast transformation using exogenous HNF1A messenger both foetal and adult connective tissue cells. Using lentivi- RNA (mRNA) [57]. Within the same year, Du et al. derived ruses as a vector for expressing transcription factors, hepato- HLCs from human fibroblasts, with the generated hepatocytes cyte nuclear factor 1 homeobox alpha (HNF1A), HNF4A and possessing phase I/II/III drug metabolic activity comparable to FOXA3, successful fibroblast to HLC transformation has primary human hepatocytes [58]. Fibroblast-derived HLC been achieved, with HNF1A being crucial for the human production requires only a single transformation step and they Table 1 Summary of selected clinical trials globally, researching the therapeutic benefits of alternative cell sources in liver disease Alternative cell sources Advantages Disadvantages Induced pluripotent stem � Patient-specific cell generation � Inadequate long-term functionality cells (iPSC) � Reduced immune response � Potential tumour formation � Disease modelling � Limitless pool of cells Fibroblasts � Patient-specific cell generation � Proliferation arrest � Reduced immune response � Residual epigenetic memory � Disease modelling � Resistant to transformation Mesenchymal stem/stromal � Patient-specific cell generation � Could potentially lose functionality cells (MSC) � Immune modulation � Easily accessible from several tissues of the body Hepatic progenitor � Naturally differentiate into new hepatocytes � Could play no role in liver regeneration cells (HPC) � Can be used to generate a number of new hepatocytes � May be involved in the progression of liver fibrosis � Considered to be involved in hepatocyte regeneration � Acquired from donor livers so may cause immune rejection � Immunosuppressive drugs would be required Amniotic epithelial � Reduced risk of tumour formation � Gene expression similar to foetal cells rather cells (hAEC) � Easily accessible than adult hepatocytes � Reduced ethical implications � Reduced immune reaction J Mol Med (2018) 96:469–481 473 can be patient-specific, reducing the chances of immune re- differentiated adult hepatic stem cells are also capable of urea jection and avoiding the use of immunosuppressive drugs [38, production and ammonium chloride metabolism [63, 64]. 49, 56]. Despite these advantageous, fibroblast-derived HLCs Unlike some other HLCs, hepatocytes formed from liver- have therapeutic limitations. They have a limited reproduc- derived progenitor cells have reached clinical application. tive capability and cannot be used for repeat infusions in a Sokal et al. transplanted HPCs in a 3-year-old female patient single patient. Furthermore, human fibroblasts are resistant suffering with OTC deficiency. Previous transplantation of to hepatic transdifferentiation, thereby creating an addition- cryopreserved hepatocytes failed to improve the patient’s al barrier when generating HLCs [56]. Hepatocytes gener- symptoms. Fourteen weeks post-infusion, biopsies showed ated from reprogrammed fibroblasts may still retain epige- 3% presence of donor cells and the patient showed some func- netic memory from the fibroblast cell of origin. This creates tional improvement with a reduction in disease-related anorex- limitations when choosing fibroblasts for hepatic transfor- ia. Unfortunately, 6 months post-infusion, the child underwent mation, as cells with significant epigenetic differences to OLT and later died. These results suggest that HPCs could play hepatocytes may be further resistant to reprogramming a role in treatment of metabolic liver disease; however, longer- and reduced functionality [59]. scale clinical trials are required to assess their full potential [65]. Hepatic differentiation of amniotic epithelial cells Human bile duct cells It is also possible to generate hepatocytes from amniotic epi- In addition to HPCs, mature hepatocytes can be derived from a thelial cells. These cells have stem cell markers such as OCT- number of other resident cell types within the liver. Huch et al. 4, Nanog, SOX-2 and Rex-1, and as they do not have telome- established a protocol differentiating primary human bile duct rase reverse transcriptase, they show a stable phenotype with- cells (EpCAM ) into genetically stable functional HLCs in both out the risk of tumorigenic potential [60]. Such cells have in vitro and in vivo transplantations. Organoids were formed minimal ethical implications, and there is no shortage of pla- using ductal cells, and using medium consisting of BMP7, EGF cental tissue from which to isolate the cells. Following culture and HGF, successful hepatic differentiation was achieved. in Matrigel® or liver-derived ECM, these cells had albumin Newly formed HLCs demonstrated albumin production, and CYP3A4, 3A7, 2B6 and 2D6 mRNA levels which in- CYP3A3/4/5 activity and bile acid secretion. Furthermore, creased over time with a peak at day 21 [61]. Following trans- organoids successfully engrafted into Balb/c nude mice with plantation into the SCID mouse, genes were expressed for induced liver damage, sustaining albumin and α-1-antitrypsin human cytochrome p450 genes, metabolic enzymes and levels for up to 120 days in two out of five recipient mice. hepatocyte-enriched transcription factors and plasma proteins Debate remains over the genetic stability of fibroblasts and 6 months post-transplantation. It has now been suggested that iPSCs as cell sources for HLCs. The expandable nature and hepatic differentiation of amniotic epithelial cells (hAECs) genetic stability of HLCs derived from human bile ductal cells represent a promising non-controversial, unlimited source of makes them a desirable alternate cell source [66]. cells for liver-based metabolic diseases. Chemically induced liver progenitors The regenerative capacity of resident liver cell hepatic progenitor cells Recently, it has been shown that mature hepatocytes convert to HLCs during chronic liver injury [67]. Katsuda et al. showed Hepatic progenitor cells (HPCs), also known as oval cells, are that a cocktail of small molecules Y-27632, A-83-01 and believed to differentiate into mature hepatocytes or CHIR99021 could contribute to the induction and maintenance cholangiocytes, upon liver damage and help in tissue restora- of bipotent chemically induced liver progenitor cells (CLiPs). tion. HGF and EGF are critical in inducing the transformation of These cells could either be differentiated into mature hepato- HPC into hepatocytes. HGF activates the MET receptor, which cytes or biliary epithelial cells. Rat CLiPs were capable of further upregulates the expressions of AKT and STAT3 driving repopulating immunodeficient mice with chronic liver injury. hepatic transformation. A lack of MET receptors completely Albumin levels were used to assess liver functionality, which attenuates HPC to hepatocyte differentiation even in the pres- showed a consistent increase up until 8 weeks post-transplanta- ence of EGFR. However, EGFR-null HPCs were still able to tion. Immunohistochemistryshowedthatupto75–90% of the sufficiently transform into hepatocytes with MET alone [62]. mouse liver had been replaced by rat hepatocytes, demonstrating Zhangetal. establishedinvitrogenerationofHLCsfrom a selective proliferative advantage for the healthy donor cells. human foetal HPCs, under the influence of oncostatin M Mouse ductal structures also showed CLiPs-derived cell re- (OSM), DEX and HGF. These newly differentiated hepatocytes placement, displaying the biopotency of the lineage. If similar have functional glycogen storage, albumin secretion and cyto- protocols could be established using human hepatocytes, this chrome p450 activity with Khuu et al., suggesting that in vitro offers an additional source for HLCs for use in HT. 474 J Mol Med (2018) 96:469–481 Furthermore, the bipotent properties of CLiPs suggest that they to promote liver repair, MSC-mediated transfer of mitochondria could be used to tackle diseases related to the biliary tree as well by tunnelling nanotubules and by MSC-mediated transfer of as the liver [68]. proteins, RNA, hormones and chemicals by extracellular vesi- cles such as exosomes or microvesicles [75]. MSC conditioned medium (MSC-CM) can play an impor- The role of mesenchymal stem/stromal cells tant role in attenuating liver disease with a wide range of soluble in liver-based regenerative medicine factors thought to be present within MSC-CM [76]. Interleukin- 6 secreted by MSCs reduces apoptosis in liver injury [77]. Mesenchymal stem/stromal cells (MSCs) have been investi- Furthermore, MSC secreted TGF-β and nerve growth factor gated as another cell source for hepatocyte differentiation but resulted in apoptosis of hepatic stellate cells, a hallmark of liver with limited and controversial results. More promising is their fibrosis [78, 79]. Huang et al. showed that mice with fulminant immunogenic effect, and now, MSCs are being investigated as hepatic failure (FHF) and chronic liver failure treated with an immunomodulatory therapy to treat liver a number of dif- MSCs or MSC-CM displayed reduced liver pathology. Only ferent liver diseases. MSC treatment of FHF mice showed great reduction in pro- MSCs can be isolated from various tissues, such as bone inflammatory T helper-1/17 cells and upregulation of T regula- marrow, adipose tissue and the umbilical cord. BMP and fi- tory cells. This indicates that direct presence of MSCs is re- broblast growth factor (FGF) induction lead to the differenti- quired to induce complete immunomodulatory effects [80]. ation of MSCs to the hepatic lineage, with dexamethasone In addition to soluble factors present in MSC-CM, recently, (DEX) and IL-16 inducing hepatic maturation. Single-step exosomes have been identified as an important component procedures are also commonly used with HGF and epidermal that may promote hepatic regeneration. Tan et al. (2014) growth factor (EGF). Transformed cells generated from this showed that CCL4-induced liver injury reduced AST and procedure usually exhibit functional hepatic properties 2– ALT levels and decreased the number of necrotic cells in mice 3 weeks post-culturing but do lose functional capabilities that were treated with MSC-derived exosomes. Furthermore, when cultured for prolonged periods [69]. Furthermore, it is proliferation of hepatocytes was greater, which was associated still debated whether MSC-derived hepatocytes are able to with increased expression of proliferating cell nuclear antigen efficiently re-populate a host liver to provide adequate func- [81]. The authors suggest that MSC-derived exosomes may tion and clinical application is still in its infancy [70]. Perhaps have a therapeutic potential in toxic liver injury. It has also of greater potential is the immunomodulatory effect of MSCs. been suggested that tunnelling nanotubules can form between MSCs play a key role in immune modulation due to their lack cells that act as a transport network, allowing the transport of of MHC-I/MHC-II receptors and are unlikely to trigger a T mitochondria and lysosomal vesicles [82]. Currently, the transfer of mitochondria through tunnelling nanotubules from cell response. Furthermore, MSCs are considered to reduce T cell proliferation and cytotoxicity, as well as improving liver umbilical cord-derived MSCs to hepatocytes is being investi- injury and enhancing liver regeneration [71, 72]. This immune gated as a mechanism for their increased survival and function evasion capability has resulted in MSCs adopting the title of and may play a role in liver regeneration [83]. Bimmunoprivileged^ or Bimmunotolerant^ cells [73]. Thus, MSCs represent an ideal cell source for liver regeneration- MSCs could be used in conjunction with hepatocytes dur- based medicine due to their easily accessible source, their ing HT to increase engraftment and reduce the immune immunomodulatory properties and their potential of response. Hwang et al. showed that intrasplenic transplan- transdifferentiating into hepatocytes. tation of MSC-derived HLCs restored liver functionality in rat models with thioacetamide-induced liver cirrhosis. Naïve implanted MSCs firstly transdifferentiated into he- Current clinical trials using cell therapy patic oval cells and later into HLCs. The presence of newly for liver-based diseases formed HLCs reduced inflammation, reversed fibrosis and repaired damaged hepatocytes. The exact mechanism by There are now multiple phase I/II and III clinical trials using which MSCs induce hepatic recovery is unclear, but the different types of stem cells to improve a number of liver authors suggest that activation of humoural factors could diseases including cirrhosis, liver failure and liver-based met- contribute to liver regeneration [74]. abolic disorders. In liver cirrhosis and end-stage liver disease, reports have proposed that MSCs can replace hepatocytes in Mechanisms of MSCs in liver regeneration the injured liver, stimulating liver regeneration (Table 2). Shi et al. (2012) showed that transfusion of umbilical cord- Recently, it has been suggested that MSCs modulate liver fail- MSC (UC-MSC) into 24 patients with acute-on-chronic liver ure by several mechanisms including differentiation of MSCs failure showed marked increase in liver functionality when to replace damaged cells, secretion of soluble factors by MSCs compared to the control of 19 patients transfused with saline. J Mol Med (2018) 96:469–481 475 Table 2 A summary of the advantages and disadvantages of various cell sources which can be used to generate induced hepatocytes for hepatocyte transplantation Study name Cell source Condition Intervention Primary outcome Study Location Start and References phase end date Umbilical cord Umbilical Liver failure Conventional treatment Survival rate and Phase I Department of Infectious November 2012– [84] mesenchymal stem mesenchymal only (antiviral drugs, time (time frame and II Diseases, The Third March 2015 cell transplantation stem cell lowering aminotransferase 48 weeks) Affiliated Hospital of combined with (UC-MSC) and jaundice medicine) Sun Yat-Sen University plasma exchange Conventional treatment Guangzhou, for patients with plus UC-MSC Guangdong China liver failure transplantation (via peripheral vein slowly for 30 min 1 × 10 /kg, once a week, four times) or plasma exchange (2000 mL every 3 days, three times) or both Safety and efficacy of Umbilical Liver cirrhosis Conventional treatment One-year survival Phase I Xijing Hospital of September 2012– [85] human umbilical mesenchymal End-stage liver or UC-MSC rate (time frame and II Digestive Disease September cord-derived stem cell disease transplantation 1-year treatment) Xi’an, Shaanxi, 2015 mesenchymal stem (UC-MSC) (1 × 10 cells/kg via China cells for treatment of hepatic artery) HBV-related liver cirrhosis Phase II safety study Human liver-derived Acute-on-chronic Low-dose cohort—two dose Occurrence of adverse Phase II Hȏpital Erasme, December 2016– [86] of two dose regimens of mesenchymal liver failure regimens of HepaStem will events (AEs) up to Brussels, Belgium September HepaStem in patients stem cell be given, differing in cell day28of the active UZ Antwerpen, 2018 with ACLF (HEP101) (HepaStem) quantity per infusion. The study period (time Edegem, Belgium low dose regimen will be frame up to 28 days KU Leuven, Leuven, given to the first cohort post-first infusion day) Belgium (first six patients included CHU de Liège, in the study). Liège, Beligium High-dose cohort—given to Cliniques St. Luc, the second cohort after Woluwe-Saint evaluation of the safety Lambert, Belgium of the first cohort Hȏspital Beaujon, (stepwise approach) Clichy, France Hȏpital de la Croix Rousse, Lyon, France HȏpitalPaulBrousse, Villejuf, France Bone marrow stem cells Bone marrow Familial Bone marrow stem cell Serum cholesterol Phase I Digestive Disease June 2007– [87] as a source of stem cells hypercholesterolemia transplantation. and LDL level Research Center, June 2008 8 9 allogenic hepatocyte 6× 10 to 1 × 10 cells (time frame Shariati Hospital, transplantation in infused through the portal 6months) North Kargar Ave., homozygous familial vein over 30 min, Tehran, Iran, Islamic hypercholesterolemia done once Republic 476 J Mol Med (2018) 96:469–481 Table 2 (continued) Study name Cell source Condition Intervention Primary outcome Study Location Start and References phase end date Study to evaluate the Human liver-derived Urea cycle HepaStem administered in Efficacy as determined Phase II Cliniques Universitaires October 2014– [88] efficacy of HepaStem mesenchymal disorders maximum four infusion by de novo ureagenesis Saint-Luc, Brussels, March 2017 in urea cycle disorders stem cell days, spread over (C13 tracer method) Belgium, H pital of paediatric patients (HepaStem) 8 weeks, with 2/3-week (time frame 6 months Jeanne de Flandre, (HEP002) interval between post-first infusion day) CHRU Lille, Lille, infusions. Target total France dose 5 × 10 /kg body Instytut–Pomnik weight Centrum Zdrowia Dziecka, Warszawa, Poland Hospital Materno Infatil de Badajoz, Badajoz, Spain Hospital Universitari Vall d’Hebron de Barcelona, Barcelona, Spain Hospital Materno Infantil de Málaga, Málaga, Spain Safety and tolerance of Bone marrow- Paediatric liver Two doses of 1 × 10 Number of participants Phase I University Children’s July 2013– [89] immunomodulating derived transplantation MSCs/kg body with MYSTEP-score Hospital, Tubingen, Januray 2019 therapy with MSCs weight grade 3 and grade 2 Germany donor-specific MSC (toxicity of MSC in paediatric liver-donor infusion), number of liver transplantation participants with (MYSTEP1) occurrence of any severe adverse events, graft function after liver transplantation, number of participants with abnormal liver tests) Therapeutic strategy and Mesenchymal Liver Six doses of 1 × 10 /kg Efficacy 1-year graft Phase I The Third Affiliated February 2014– [90] the role of stem cells transplantation body weight MSCs survival rate Hospital, Sun Yat-Sen March 2017 mesenchymal stromal are given, intravenously University, Guangzhou, cells for ABO Guangdong, China incompatible liver transplantation Human mesenchymal Umbilical cord Acute-on-chronic Conventional treatment Liver functionality Phase I Beijing 302 Hospital March 2009– [91] stem cell transfusion mesenchymal liver failure and 0.5 × 10 /kg body tested over 48 weeks and II Beijing, Beijing, March 2014 is safe and improves stem cell weight UC-MSCs are 72-week survival rate China liver function in (UC-MSC) given, intravenously at acute-on-chronic baseline, 4 weeks, and liver failure patients 8weeks J Mol Med (2018) 96:469–481 477 Table 2 (continued) Study name Cell source Condition Intervention Primary outcome Study Location Start and References phase end date Conventional treatment and saline was used for the control group Safety study of Human liver-derived Urea cycle HepaStem low dose Safety of HepaStem in Phase I Saint Luc University March 2012– [92] HepaStem for the mesenchymal disorders 12.5 × 10 /kg body paediatric patients and II Hospital, Brussels, April 2015 treatment of urea cycle stem cell Crigler Najjar weight suffering from urea Belgium disorders (UCD) and (HepaStem) syndrome HepaStem intermediate cycle disorder and Universitair Ziekenhuis Crigler-Najjar dose 50 × 10 /kg body Crigler-Najjar (US) Antwerpen, syndrome (CN) weight syndrome Edegem Belgium (HEP001) HepaStem high dose CHU Bicetre, Le Kremlin 200 × 10 /kg body Bic tre Cedex, France weight H pital Jeanne de Flandre, CHRU Lille, Lille Cedex, France H pital des Enfants, CHU de Toulouse, Toulouse, Cedex 9, France Rambam Medical Center, Meyer Children’s Hospital, Haifa, Israel Hadassah Ein-Kerem Medical Center of Israel, Jerusalem, Israel Schneider Children’sMedical Center of Israel, Petach Tikva, Israel Ospedale Pediatrico BambinoGesudiRoma, Roma, Italy Birmingham Children’s Hospital London, London, United Kingdom Macrophage Autologous Liver cirrhosis Autologous activated Liver function Phase I Edinburgh Royal Infirmary August 2016– [93] therapy for liver macrophages macrophages infusion (MELD score) and II Little France Crescent August 2021 cirrhosis via peripheral vein for at 3 months Edinburgh EH16 4SA 30 min. United Kingdom Standard medical care used as the control Information from Clincialtrials.gov [80] 478 J Mol Med (2018) 96:469–481 Patients were monitored over 48 weeks, with the treatment Another promising area could be the use of macrophage group showing an increase in albumin secretion, platelet count therapy to treat liver disease. Macrophages reduce scar tissue and a reduced end-stage liver disease (MELD) score. and stimulate the HPCs to expand and differentiate into mature Furthermore, survival rate after 72 weeks was also higher in hepatocytes. Thomas et al. showed that bone marrow-derived the treatment group compared to the control, with 20.8 and macrophages (BMM) administered to mice with advanced liver 47.4% mortality rate, respectively. The author suggests that fibrosis resulted in a degradation of fibrillar collagen and re- although the mechanism of improved liver function may be duced fibrogenesis. There was also upregulation of the liver unclear, in vivo differentiation of UC-MSC into hepatocytes progenitor cell mitogen tumour necrosis factor-like weak in- is unlikely due to the short period of hepatic recovery and with ducer of apoptosis that was associated with an expansion of only one treatment patient showing increased alpha-fetoprotein the progenitor cell compartment [97]. There are ongoing clini- levels. It is more likely that soluble factors produced by MSCs cal trials to assess the role macrophage therapy could play in may enhance liver revascularization and proliferation [95]. liver cirrhosis [93]. Significant advances have been made to One study has suggested that plasma exchange (PE) helps translate the use of stem cells to promote liver regeneration promote liver regeneration and recovery, leading to UC-MSC and mature hepatic differentiation into clinical use. Currently, differentiation into HLCs. A phase I/II clinical trial is now in most trials are in early phase I/II and results demonstrating the progress, transplanting UC-MSCs into patients with liver fail- efficacy of these techniques are yet to be published. In the near ure. Patients received either conventional treatment (anti-viral future, the full potential of stem cells for liver regeneration in drugs) with UC-MSCs and/or PE treatment, and survival rates patients with liver disease may be better established. were assessed at 48 weeks [84]. For patients with acute-on- chronic liver failure, Promethera Biosciences have developed a product known as HepaStem, which are MSCs that have the Conclusion potential to differentiate into HLCs. A phase IIa clinical trial is now in progress, transplanting these cells via IV injection to HT offers an alternative therapy to OLT with the aim of treating establish the safety and biological efficacy of these cells. liver-based metabolic diseases or ALF. Advances in liver cell Bilirubin, creatinine, INR and albumin values are being therapy are being researched to overcome the obstacles associ- assessed at day 28, 2 months and 1 year post-infusion. In addi- ated with HT, particularly the shortage of healthy donor hepa- tion to using stem cells for liver failure, HLCs are now being tocytes. Although HLCs are promising, no alternative cell used for clinical HT to replace primary hepatocytes in patients source can yet replace the functionality and efficacy of primary with liver-based metabolic disorders. Bone marrow-derived human hepatocytes. MSCs likely hold the greatest attribute as MSCs transdifferentiated into hepatocytes have been immunomodulators, and co-culturing with mature donor hepa- transplanted via the portal vein into patients with familial hy- tocytes. More clinical trials assessing the safety and efficacy of percholesterolemia. Serum cholesterol/LDL levels were HLCs is pivotal before they can be considered as a reliable cell assessed after 6 months to determine the efficacy of the tech- source. In the future, this may allow for liver-based diseases to nique. Furthermore, HepaStem cells are also being used to treat be effectively treated without the need for OLT. patients suffering from urea cycle disorders. Ureagenesis, am- monia values and amino acid levels are being monitored as well Compliance with ethical standards as behaviour, cognitive skills and health-related quality of life indicators for up to 12 months post-infusion [96]. Conflict of interest The authors declare that they have no conflict of MSCs are also being used clinically for immunomodulating interest. therapy in many liver-based applications. One trial is currently Open Access This article is distributed under the terms of the Creative investigating the use of MSCs to promote allograft tolerance Commons Attribution 4.0 International License (http:// and reduce the toxicity that results from exposure to calcineurin creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appro- inhibitors. Paediatric patients receiving a liver transplantation priate credit to the original author(s) and the source, provide a link to the undergo IV injection of bone marrow-derived MSCs. MSC Creative Commons license, and indicate if changes were made. toxicity is being monitored as well as graft function measured by aminotransferase and gamma glutamyl transferase activity, bilirubin, albumin and INR and the individual need for immu- References nosuppressive medication. In addition, MSCs are being used as immunomodulators in ABO-incompatible liver transplantation. 1. 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In: Clinicaltrials.gov http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Molecular Medicine Springer Journals

Hepatocyte transplantation and advancements in alternative cell sources for liver-based regenerative medicine

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Springer Journals
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Copyright © 2018 by The Author(s)
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Biomedicine; Molecular Medicine; Human Genetics; Internal Medicine
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0946-2716
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1432-1440
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10.1007/s00109-018-1638-5
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Abstract

Human hepatocyte transplantation has been actively perused as an alternative to liver replacement for acute liver failure and liver-based metabolic defects. Current challenges in this field include a limited cell source, reduced cell viability following cryopreservation and poor engraftment of cells into the recipient liver with consequent limited life span. As a result, alternative stem cell sources such as pluripotent stem cells, fibroblasts, hepatic progenitor cells, amniotic epithelial cells and mesenchymal stem/stromal cells (MSCs) can be used to generate induced hepatocyte like cells (HLC) with each technique exhibiting advantages and disadvantages. HLCs may have comparable function to primary human hepatocytes and could offer patient-specific treatment. However, long-term functionality of transplanted HLCs and the potential oncogenic risks of using stem cells have yet to be established. The immunomodulatory effects of MSCs are promising, and multiple clinical trials are investigating their effect in cirrhosis and acute liver failure. Here, we review the current status of hepatocyte transplantation, alternative cell sources to primary human hepatocytes and their potential in liver regeneration. We also describe recent clinical trials using hepatocytes derived from stem cells and their role in improving the phenotype of several liver diseases. . . . . Keywords Hepatocyte transplantation Induced pluripotent stem cells Fibroblast Mesenchymal stem/stromal cell Hepatic progenitor cells Liver regeneration Abbreviations fVII Factor VII ALF Acute liver failure FGF Fibroblast growth factor BMM Bone marrow-derived macrophages FHF Fulminant hepatic failure BMP4 Bone morphogenetic protein 4 hAECs Hepatic differentiation of amniotic CN-1 Crigler-Najjar syndrome type 1 epithelial cells CYP Cytochrome p450 HPCs Hepatic progenitor cells DEX Dexamethasone HNF1A Hepatocyte nuclear factor 1 EGF Epidermal growth factor homeobox alpha HT Hepatocyte transplantation HGF Hepatocyte-growth factor HLCs Hepatocyte-like cells Siddharth Sinha and Charlotte A Lee are joint first authors iPSCs Induced pluripotent stem cells PE Plasma exchange * Anil Dhawan Anil.dhawan@kcl.ac.uk EpCAM Primary human bile duct cells CLiPs Proliferative bipotent cells Dhawan Lab, Institute of Liver Studies, King’s College London at MSCs Mesenchymal stem cells King’s College Hospital NHS Foundation trust, London, UK MSC-CM MSC-conditioned medium Paediatric Liver GI and Nutrition Centre, King’s College London at OSM Oncostatin M King’s College Hospital NHS Foundation Trust, London, UK OTC Ornithine transcarbamylase deficiency OLT Orthotopic liver transplantation UC-MSC Umbilical cord–mesenchymal stem cell 470 J Mol Med (2018) 96:469–481 Introduction the primary long-term treatment is liver transplantation, the lack of an appropriately sized, blood matched donor organ The liver is responsible for a diverse range of functions within can lead to irreversible neurological disability. There are re- the body ranging from xenobiotic metabolism to plasma pro- ports of 8 patients that have received between 1.4 and 7.5 tein synthesis [1]. However, the liver can become susceptible billion hepatocytes, which showed up to a 50% reduction in to acute failure (ALF) due to viral infection or drug effect bilirubin and UGT activity and a decrease in the need for amongst other aetiologies [2]. Liver-based metabolic disor- phototherapy. The majority of these patients went onto receive ders that are caused by single-gene defects can result in a lack an OLT within a year as the effects of HT were not long of specific liver-based enzyme function which may result in lasting, with one patient avoiding OLT for 4 years [16–21]. damage to other organs most often irreversible neurodisability Several urea cycle defects have also been shown to benefit [3]. Orthotopic liver transplantation (OLT) is currently the from hepatocyte infusions. This includes ornithine only viable treatment for severe ALF and certain liver-based transcarbamylase deficiency (OTC), which is an X-linked ge- metabolic disorders [2, 4, 5]. Liver replacement is not futuris- netic condition affecting males that causes hyperammonemia tic for liver-based metabolic defects where gene therapy and can also lead to neurological implications including devel- would be ideal. Notably, a shortage in healthy donor livers opmental delay and learning disabilities. HT led to decreased has led to research into alternative treatment options [6]. ammonia levels and increased urea production in six of these Hepatocyte transplantation (HT), where liver cells may pro- patients, with four receiving an OLT within 7 months and two vide a potential alternative to OLT or act as a bridge until a reported mortalities [14, 22–25]. Other metabolic liver diseases suitable organ becomes available, has been demonstrated, shown to improve following HT include familial hypercholes- with over 100 cases published worldwide showing the safety terolemia where there was up to a 20% decrease in LDL in and preliminary efficacy of the technique [7, 8]. three patients, fVII deficiency where there was a 70% decrease This review will focus on the current status of HT, alternate in the need for recombinant fVII for 6 months and ASL defi- cell sources to primary human hepatocytes and alternate ciency where the was a decrease in ammonia production and methods of liver regeneration which can be used either inde- increased psychomotor abilities [26–28]. Nevertheless, the pendently or in combination with HT. long-term benefits of HT for the treatment of patients with liver-based metabolic disease have yet to be shown. HT can also be used in patients with ALF. Although liver The current status of hepatocyte transplantation is the primary treatment, paediatric patients transplantation may die whilst waiting for an appropriately size and blood- matched liver, with no other treatment being shown to im- prove survival [29]. Furthermore, existing listing criteria are HT is a less invasive alternative to OLT. Hepatocytes are iso- lated from donor livers that have been rejected for organ trans- not robust in terms of accurately predicting death and survival; plantation in view of prolonged warm or cold ischaemia times, hence, some patients receive a liver transplantation, even if mild steatosis or aberrant anatomy. Hepatocytes isolated from their liver may spontaneously recover, leading to unnecessary one donor liver can yield a high quantity of cells and be used surgeries with life-long immunosuppression. Thirty-seven pa- to treat multiple patients [9, 10]. The ability to cryopreserve tients with acute liver failure have received human hepato- these cells is one of the major advantages of HT, potentially cytes for both drug induced, viral and idiopathic ALF. Ten providing an off-the-shelf treatment and enabling cells of all of these patients underwent intraportal hepatocyte infusions, blood groups to be immediately available for emergency with two making a full recovery without the need for OLT and cases, which is particularly important for patients with ALF. three were successfully bridged to transplantation [15, 30–33]. Several routes of infusion for cell transplantation have been However, there is a high risk associated with placing described including intraportal, intrasplenic and intraperitone- intrahepatic invasive catheters in coagulopathic patients. As al. Depending on the age of the patient, infusion of hepato- an alternative, it is possible to encapsulate hepatocytes in al- cytes into the portal vein can be undertaken via the umbilical ginate microbeads made from purified alginate using an vein under fluoroscopy to avoid a patent ductus venous. In encapsulator [34]. The semi-permeable membrane within the older children, radiological or surgical catheters are required microbeads allows exchange of metabolites, maintaining syn- using radiological guidance [11–13]. thetic detoxification function, whilst also protecting against Hepatocyte transplantation can be used to treat patients immunocompetent cells by preventing the entry of antibodies with liver-based metabolic diseases and ALF [7, 14, 15]. [35, 36]. Hepatocyte-containing microbeads are transplanted Metabolic liver diseases treated using HT include Crigler- into the intraperitoneal cavity. The transplanted hepatocytes Najjar syndrome type 1 (CN-1), urea cycle defects and factor can assist the necessary liver functions, allowing the liver to VII (fVII) deficiency [16]. Crigler-Najjar syndrome has an recover. After full recovery, usually 1 month, the microbeads incidence of 1 in 1,000,000 births in the UK, and although can be removed using a laparoscopic peritoneal lavage. J Mol Med (2018) 96:469–481 471 Despite extensive clinical data, long-term clinical out- hepatoblast formation and hepatocyte-like differentiation, re- comes of HT have yet to be established with any type of spectively. Si-Tayeb et al. showed that iPSC-derived HLCs liver-based metabolic disease or ALF. There are still many displayed hepatic functionality, including glycogen accumu- limitations to the technique, and current research aims to over- lation, lipoprotein uptake and urea synthesis [47]. iPSCs are come this. Firstly, the cryopreservation process requires fur- advantageous over primary human hepatocytes as generation ther optimisation with the current protocol resulting in low from somatic cells of an individual will prevent activation of viability and function of hepatocytes post-thawing. the recipient’s immune response, avoiding the use of immu- Furthermore, transplanted donor hepatocytes undergo im- nosuppressive drugs [38, 45, 48, 49]. iPSCs can proliferate mune rejection with up to 70% of engrafted cells cleared with- indefinitely, forming a limitless pool of HLCs allowing pa- in the first 24 h post-transplantation [37]. Upon transplanta- tients to receive multiple infusions if necessary [48, 50]. tion, donor hepatocytes are recognised and activate the instant iPSC-derived HLCs can also be used as a model for several blood-mediated inflammatory reaction, during which both the metabolic liver diseases including familial hypercholesterol- complement and coagulation pathways are activated. Innate emia, α -antitrypsin deficiency and glycogen storage disease immune cells such as Kupffer cells, natural killer cells and type 1a. By generating cells with similar phenotypes to those monocytes rapidly clear transplanted donor hepatocytes [38, caused by inherited diseases, it is possible to study the mech- 39]. A major limitation of HT is the lack of good quality donor anisms of dysregulated cellular functions and identify ways to organs from which to isolate cells from. Neonatal livers have treat or reverse the condition [51]. Currently, the functional been investigated as a potential cell source as they are infre- ability of HLCs is not comparable to human primary hepato- quently used for OLT due to the high incidence of hepatic cytes. Yu et al. showed that HLCs co-express alpha-fetopro- artery thrombosis [40]. However, they may be an ideal cell tein and albumin, suggesting that they are not fully mature. source for HT, with one study demonstrating their mature Levels of albumin synthesis, urea production, cytochrome function of cytochrome P450 and ureagenesis enzymes, with p450 activity and mitochondrial function are also significantly increased attachment efficiency and viability compared to he- lower than human primary hepatocytes [50]. Furthermore, patocytes isolated from adult donor livers [41, 42]. Currently, there are genetic variations in donor cells that effect the dif- the number of neonatal donations is not sufficient to maintain ferentiation propensity of iPSCs which has been attributed to an active clinical HT programme [41, 43]. As a result, there is genetic variation of the donor, cell culture conditions and a requirement to investigate alternative cell sources to resolve iPSC generation protocols [52, 53]. Following the develop- the shortage of healthy donor organs and avoid recipient im- ment of new hepatic differentiation protocols, Kajiwara et al. mune rejection (Fig. 1 and Table 1). compared 28 iPSC lines derived from different somatic cells and showed that it was the origin of the donor cells that deter- mined the variation in hepatic differentiation and not the der- Alternative cell sources for hepatocyte ivation method [53].With albumin used as a marker to assess transplantation functionality, HLCs derived from human dermal fibroblast- iPSCs and peripheral blood cell-iPSCs showed minimal vari- Induced pluripotent stem cells generated ation in hepatic differentiation from the same donor; however, from somatic cells inter-donor hepatic variation was more prominent. This cre- ates a complication when using iPSCs for therapeutic use as Induced pluripotent stem cells (iPSCs) that can be differenti- non-identical cell lines cannot guarantee the same quality of ated into hepatocyte-like cells (HLCs) are being widely ex- HLC production [48, 53]. plored as an alternative to primary human hepatocytes. These In addition, the tumorigenic potential of these cells due to cells offer an excellent alternative source of hepatocytes and the presence of oncogenes such as c-Myc may raise a safety are advantageous over primary human hepatocytes because of concern regarding the clinical application of these cells. Chen their unlimited cell source and their ability to avoid the im- et al. reported no formation of teratomas or tumours in Gunn mune system [44]. Methods to produce iPSC-derived HLCs rats transplanted with HLCs, suggesting a loss of pluripotent are well established, with most protocols using a three- characteristics within these cells [54]. Although HLCs repre- dimensional matrix such as Matrigel® to establish the gener- sent an ideal cell source for HT with no risk of rejection, work ation of hepatocytes. However 2D matrices including collagen is still ongoing to advance their functional capacity and fully have also shown successful iPSC to HLC differentiation [45, validate the safety and efficacy of using these stem cells. 46]. An effective protocol for generating HLCs has been established via the use of specific growth factors such as Fibroblasts activin A and bone morphogenetic protein 4 (BMP4), which are crucial for initiating the first step of hepatic maturation. Human fibroblasts offer another potential source of HLCs for Hepatocyte-growth factor (HGF) and oncostatin stimulate HT. Fibroblasts are connective tissue cells found in all areas of 472 J Mol Med (2018) 96:469–481 Fig. 1 Potential alternative cell sources (induced pluripotent stem cells, HPC hepatic progenitor cells, hAEC human amniotic epithelial cells, fibroblasts, mesenchymal stem/stromal cells and hepatic progenitor cells) BMP bone morphogenetic protein, OSM oncostatin M, HGF hepatic which can be used to generate hepatocytes. Gene transfer is used to growth factor, HNF1A hepatocyte nuclear factor 1 homeobox alpha, convert somatic cells to iPSCs and fibroblasts to HLCs. All other trans- HNF4A hepatocyte nuclear factor 4 alpha, FGF fibroblast growth factor, formations occur under culture conditions. HLC induced hepatocyte, EGF epidermal growth factor, Dex dexamethasone, FBS foetal bovine iPSC induced pluripotent stem cells, MSC mesenchymal stem cells, serum the human body [55]. Huang et al. established the first fibroblast reprogramming [56]. Simeonov et al. also achieved reprogramming of human fibroblasts to hepatocytes using fibroblast transformation using exogenous HNF1A messenger both foetal and adult connective tissue cells. Using lentivi- RNA (mRNA) [57]. Within the same year, Du et al. derived ruses as a vector for expressing transcription factors, hepato- HLCs from human fibroblasts, with the generated hepatocytes cyte nuclear factor 1 homeobox alpha (HNF1A), HNF4A and possessing phase I/II/III drug metabolic activity comparable to FOXA3, successful fibroblast to HLC transformation has primary human hepatocytes [58]. Fibroblast-derived HLC been achieved, with HNF1A being crucial for the human production requires only a single transformation step and they Table 1 Summary of selected clinical trials globally, researching the therapeutic benefits of alternative cell sources in liver disease Alternative cell sources Advantages Disadvantages Induced pluripotent stem � Patient-specific cell generation � Inadequate long-term functionality cells (iPSC) � Reduced immune response � Potential tumour formation � Disease modelling � Limitless pool of cells Fibroblasts � Patient-specific cell generation � Proliferation arrest � Reduced immune response � Residual epigenetic memory � Disease modelling � Resistant to transformation Mesenchymal stem/stromal � Patient-specific cell generation � Could potentially lose functionality cells (MSC) � Immune modulation � Easily accessible from several tissues of the body Hepatic progenitor � Naturally differentiate into new hepatocytes � Could play no role in liver regeneration cells (HPC) � Can be used to generate a number of new hepatocytes � May be involved in the progression of liver fibrosis � Considered to be involved in hepatocyte regeneration � Acquired from donor livers so may cause immune rejection � Immunosuppressive drugs would be required Amniotic epithelial � Reduced risk of tumour formation � Gene expression similar to foetal cells rather cells (hAEC) � Easily accessible than adult hepatocytes � Reduced ethical implications � Reduced immune reaction J Mol Med (2018) 96:469–481 473 can be patient-specific, reducing the chances of immune re- differentiated adult hepatic stem cells are also capable of urea jection and avoiding the use of immunosuppressive drugs [38, production and ammonium chloride metabolism [63, 64]. 49, 56]. Despite these advantageous, fibroblast-derived HLCs Unlike some other HLCs, hepatocytes formed from liver- have therapeutic limitations. They have a limited reproduc- derived progenitor cells have reached clinical application. tive capability and cannot be used for repeat infusions in a Sokal et al. transplanted HPCs in a 3-year-old female patient single patient. Furthermore, human fibroblasts are resistant suffering with OTC deficiency. Previous transplantation of to hepatic transdifferentiation, thereby creating an addition- cryopreserved hepatocytes failed to improve the patient’s al barrier when generating HLCs [56]. Hepatocytes gener- symptoms. Fourteen weeks post-infusion, biopsies showed ated from reprogrammed fibroblasts may still retain epige- 3% presence of donor cells and the patient showed some func- netic memory from the fibroblast cell of origin. This creates tional improvement with a reduction in disease-related anorex- limitations when choosing fibroblasts for hepatic transfor- ia. Unfortunately, 6 months post-infusion, the child underwent mation, as cells with significant epigenetic differences to OLT and later died. These results suggest that HPCs could play hepatocytes may be further resistant to reprogramming a role in treatment of metabolic liver disease; however, longer- and reduced functionality [59]. scale clinical trials are required to assess their full potential [65]. Hepatic differentiation of amniotic epithelial cells Human bile duct cells It is also possible to generate hepatocytes from amniotic epi- In addition to HPCs, mature hepatocytes can be derived from a thelial cells. These cells have stem cell markers such as OCT- number of other resident cell types within the liver. Huch et al. 4, Nanog, SOX-2 and Rex-1, and as they do not have telome- established a protocol differentiating primary human bile duct rase reverse transcriptase, they show a stable phenotype with- cells (EpCAM ) into genetically stable functional HLCs in both out the risk of tumorigenic potential [60]. Such cells have in vitro and in vivo transplantations. Organoids were formed minimal ethical implications, and there is no shortage of pla- using ductal cells, and using medium consisting of BMP7, EGF cental tissue from which to isolate the cells. Following culture and HGF, successful hepatic differentiation was achieved. in Matrigel® or liver-derived ECM, these cells had albumin Newly formed HLCs demonstrated albumin production, and CYP3A4, 3A7, 2B6 and 2D6 mRNA levels which in- CYP3A3/4/5 activity and bile acid secretion. Furthermore, creased over time with a peak at day 21 [61]. Following trans- organoids successfully engrafted into Balb/c nude mice with plantation into the SCID mouse, genes were expressed for induced liver damage, sustaining albumin and α-1-antitrypsin human cytochrome p450 genes, metabolic enzymes and levels for up to 120 days in two out of five recipient mice. hepatocyte-enriched transcription factors and plasma proteins Debate remains over the genetic stability of fibroblasts and 6 months post-transplantation. It has now been suggested that iPSCs as cell sources for HLCs. The expandable nature and hepatic differentiation of amniotic epithelial cells (hAECs) genetic stability of HLCs derived from human bile ductal cells represent a promising non-controversial, unlimited source of makes them a desirable alternate cell source [66]. cells for liver-based metabolic diseases. Chemically induced liver progenitors The regenerative capacity of resident liver cell hepatic progenitor cells Recently, it has been shown that mature hepatocytes convert to HLCs during chronic liver injury [67]. Katsuda et al. showed Hepatic progenitor cells (HPCs), also known as oval cells, are that a cocktail of small molecules Y-27632, A-83-01 and believed to differentiate into mature hepatocytes or CHIR99021 could contribute to the induction and maintenance cholangiocytes, upon liver damage and help in tissue restora- of bipotent chemically induced liver progenitor cells (CLiPs). tion. HGF and EGF are critical in inducing the transformation of These cells could either be differentiated into mature hepato- HPC into hepatocytes. HGF activates the MET receptor, which cytes or biliary epithelial cells. Rat CLiPs were capable of further upregulates the expressions of AKT and STAT3 driving repopulating immunodeficient mice with chronic liver injury. hepatic transformation. A lack of MET receptors completely Albumin levels were used to assess liver functionality, which attenuates HPC to hepatocyte differentiation even in the pres- showed a consistent increase up until 8 weeks post-transplanta- ence of EGFR. However, EGFR-null HPCs were still able to tion. Immunohistochemistryshowedthatupto75–90% of the sufficiently transform into hepatocytes with MET alone [62]. mouse liver had been replaced by rat hepatocytes, demonstrating Zhangetal. establishedinvitrogenerationofHLCsfrom a selective proliferative advantage for the healthy donor cells. human foetal HPCs, under the influence of oncostatin M Mouse ductal structures also showed CLiPs-derived cell re- (OSM), DEX and HGF. These newly differentiated hepatocytes placement, displaying the biopotency of the lineage. If similar have functional glycogen storage, albumin secretion and cyto- protocols could be established using human hepatocytes, this chrome p450 activity with Khuu et al., suggesting that in vitro offers an additional source for HLCs for use in HT. 474 J Mol Med (2018) 96:469–481 Furthermore, the bipotent properties of CLiPs suggest that they to promote liver repair, MSC-mediated transfer of mitochondria could be used to tackle diseases related to the biliary tree as well by tunnelling nanotubules and by MSC-mediated transfer of as the liver [68]. proteins, RNA, hormones and chemicals by extracellular vesi- cles such as exosomes or microvesicles [75]. MSC conditioned medium (MSC-CM) can play an impor- The role of mesenchymal stem/stromal cells tant role in attenuating liver disease with a wide range of soluble in liver-based regenerative medicine factors thought to be present within MSC-CM [76]. Interleukin- 6 secreted by MSCs reduces apoptosis in liver injury [77]. Mesenchymal stem/stromal cells (MSCs) have been investi- Furthermore, MSC secreted TGF-β and nerve growth factor gated as another cell source for hepatocyte differentiation but resulted in apoptosis of hepatic stellate cells, a hallmark of liver with limited and controversial results. More promising is their fibrosis [78, 79]. Huang et al. showed that mice with fulminant immunogenic effect, and now, MSCs are being investigated as hepatic failure (FHF) and chronic liver failure treated with an immunomodulatory therapy to treat liver a number of dif- MSCs or MSC-CM displayed reduced liver pathology. Only ferent liver diseases. MSC treatment of FHF mice showed great reduction in pro- MSCs can be isolated from various tissues, such as bone inflammatory T helper-1/17 cells and upregulation of T regula- marrow, adipose tissue and the umbilical cord. BMP and fi- tory cells. This indicates that direct presence of MSCs is re- broblast growth factor (FGF) induction lead to the differenti- quired to induce complete immunomodulatory effects [80]. ation of MSCs to the hepatic lineage, with dexamethasone In addition to soluble factors present in MSC-CM, recently, (DEX) and IL-16 inducing hepatic maturation. Single-step exosomes have been identified as an important component procedures are also commonly used with HGF and epidermal that may promote hepatic regeneration. Tan et al. (2014) growth factor (EGF). Transformed cells generated from this showed that CCL4-induced liver injury reduced AST and procedure usually exhibit functional hepatic properties 2– ALT levels and decreased the number of necrotic cells in mice 3 weeks post-culturing but do lose functional capabilities that were treated with MSC-derived exosomes. Furthermore, when cultured for prolonged periods [69]. Furthermore, it is proliferation of hepatocytes was greater, which was associated still debated whether MSC-derived hepatocytes are able to with increased expression of proliferating cell nuclear antigen efficiently re-populate a host liver to provide adequate func- [81]. The authors suggest that MSC-derived exosomes may tion and clinical application is still in its infancy [70]. Perhaps have a therapeutic potential in toxic liver injury. It has also of greater potential is the immunomodulatory effect of MSCs. been suggested that tunnelling nanotubules can form between MSCs play a key role in immune modulation due to their lack cells that act as a transport network, allowing the transport of of MHC-I/MHC-II receptors and are unlikely to trigger a T mitochondria and lysosomal vesicles [82]. Currently, the transfer of mitochondria through tunnelling nanotubules from cell response. Furthermore, MSCs are considered to reduce T cell proliferation and cytotoxicity, as well as improving liver umbilical cord-derived MSCs to hepatocytes is being investi- injury and enhancing liver regeneration [71, 72]. This immune gated as a mechanism for their increased survival and function evasion capability has resulted in MSCs adopting the title of and may play a role in liver regeneration [83]. Bimmunoprivileged^ or Bimmunotolerant^ cells [73]. Thus, MSCs represent an ideal cell source for liver regeneration- MSCs could be used in conjunction with hepatocytes dur- based medicine due to their easily accessible source, their ing HT to increase engraftment and reduce the immune immunomodulatory properties and their potential of response. Hwang et al. showed that intrasplenic transplan- transdifferentiating into hepatocytes. tation of MSC-derived HLCs restored liver functionality in rat models with thioacetamide-induced liver cirrhosis. Naïve implanted MSCs firstly transdifferentiated into he- Current clinical trials using cell therapy patic oval cells and later into HLCs. The presence of newly for liver-based diseases formed HLCs reduced inflammation, reversed fibrosis and repaired damaged hepatocytes. The exact mechanism by There are now multiple phase I/II and III clinical trials using which MSCs induce hepatic recovery is unclear, but the different types of stem cells to improve a number of liver authors suggest that activation of humoural factors could diseases including cirrhosis, liver failure and liver-based met- contribute to liver regeneration [74]. abolic disorders. In liver cirrhosis and end-stage liver disease, reports have proposed that MSCs can replace hepatocytes in Mechanisms of MSCs in liver regeneration the injured liver, stimulating liver regeneration (Table 2). Shi et al. (2012) showed that transfusion of umbilical cord- Recently, it has been suggested that MSCs modulate liver fail- MSC (UC-MSC) into 24 patients with acute-on-chronic liver ure by several mechanisms including differentiation of MSCs failure showed marked increase in liver functionality when to replace damaged cells, secretion of soluble factors by MSCs compared to the control of 19 patients transfused with saline. J Mol Med (2018) 96:469–481 475 Table 2 A summary of the advantages and disadvantages of various cell sources which can be used to generate induced hepatocytes for hepatocyte transplantation Study name Cell source Condition Intervention Primary outcome Study Location Start and References phase end date Umbilical cord Umbilical Liver failure Conventional treatment Survival rate and Phase I Department of Infectious November 2012– [84] mesenchymal stem mesenchymal only (antiviral drugs, time (time frame and II Diseases, The Third March 2015 cell transplantation stem cell lowering aminotransferase 48 weeks) Affiliated Hospital of combined with (UC-MSC) and jaundice medicine) Sun Yat-Sen University plasma exchange Conventional treatment Guangzhou, for patients with plus UC-MSC Guangdong China liver failure transplantation (via peripheral vein slowly for 30 min 1 × 10 /kg, once a week, four times) or plasma exchange (2000 mL every 3 days, three times) or both Safety and efficacy of Umbilical Liver cirrhosis Conventional treatment One-year survival Phase I Xijing Hospital of September 2012– [85] human umbilical mesenchymal End-stage liver or UC-MSC rate (time frame and II Digestive Disease September cord-derived stem cell disease transplantation 1-year treatment) Xi’an, Shaanxi, 2015 mesenchymal stem (UC-MSC) (1 × 10 cells/kg via China cells for treatment of hepatic artery) HBV-related liver cirrhosis Phase II safety study Human liver-derived Acute-on-chronic Low-dose cohort—two dose Occurrence of adverse Phase II Hȏpital Erasme, December 2016– [86] of two dose regimens of mesenchymal liver failure regimens of HepaStem will events (AEs) up to Brussels, Belgium September HepaStem in patients stem cell be given, differing in cell day28of the active UZ Antwerpen, 2018 with ACLF (HEP101) (HepaStem) quantity per infusion. The study period (time Edegem, Belgium low dose regimen will be frame up to 28 days KU Leuven, Leuven, given to the first cohort post-first infusion day) Belgium (first six patients included CHU de Liège, in the study). Liège, Beligium High-dose cohort—given to Cliniques St. Luc, the second cohort after Woluwe-Saint evaluation of the safety Lambert, Belgium of the first cohort Hȏspital Beaujon, (stepwise approach) Clichy, France Hȏpital de la Croix Rousse, Lyon, France HȏpitalPaulBrousse, Villejuf, France Bone marrow stem cells Bone marrow Familial Bone marrow stem cell Serum cholesterol Phase I Digestive Disease June 2007– [87] as a source of stem cells hypercholesterolemia transplantation. and LDL level Research Center, June 2008 8 9 allogenic hepatocyte 6× 10 to 1 × 10 cells (time frame Shariati Hospital, transplantation in infused through the portal 6months) North Kargar Ave., homozygous familial vein over 30 min, Tehran, Iran, Islamic hypercholesterolemia done once Republic 476 J Mol Med (2018) 96:469–481 Table 2 (continued) Study name Cell source Condition Intervention Primary outcome Study Location Start and References phase end date Study to evaluate the Human liver-derived Urea cycle HepaStem administered in Efficacy as determined Phase II Cliniques Universitaires October 2014– [88] efficacy of HepaStem mesenchymal disorders maximum four infusion by de novo ureagenesis Saint-Luc, Brussels, March 2017 in urea cycle disorders stem cell days, spread over (C13 tracer method) Belgium, H pital of paediatric patients (HepaStem) 8 weeks, with 2/3-week (time frame 6 months Jeanne de Flandre, (HEP002) interval between post-first infusion day) CHRU Lille, Lille, infusions. Target total France dose 5 × 10 /kg body Instytut–Pomnik weight Centrum Zdrowia Dziecka, Warszawa, Poland Hospital Materno Infatil de Badajoz, Badajoz, Spain Hospital Universitari Vall d’Hebron de Barcelona, Barcelona, Spain Hospital Materno Infantil de Málaga, Málaga, Spain Safety and tolerance of Bone marrow- Paediatric liver Two doses of 1 × 10 Number of participants Phase I University Children’s July 2013– [89] immunomodulating derived transplantation MSCs/kg body with MYSTEP-score Hospital, Tubingen, Januray 2019 therapy with MSCs weight grade 3 and grade 2 Germany donor-specific MSC (toxicity of MSC in paediatric liver-donor infusion), number of liver transplantation participants with (MYSTEP1) occurrence of any severe adverse events, graft function after liver transplantation, number of participants with abnormal liver tests) Therapeutic strategy and Mesenchymal Liver Six doses of 1 × 10 /kg Efficacy 1-year graft Phase I The Third Affiliated February 2014– [90] the role of stem cells transplantation body weight MSCs survival rate Hospital, Sun Yat-Sen March 2017 mesenchymal stromal are given, intravenously University, Guangzhou, cells for ABO Guangdong, China incompatible liver transplantation Human mesenchymal Umbilical cord Acute-on-chronic Conventional treatment Liver functionality Phase I Beijing 302 Hospital March 2009– [91] stem cell transfusion mesenchymal liver failure and 0.5 × 10 /kg body tested over 48 weeks and II Beijing, Beijing, March 2014 is safe and improves stem cell weight UC-MSCs are 72-week survival rate China liver function in (UC-MSC) given, intravenously at acute-on-chronic baseline, 4 weeks, and liver failure patients 8weeks J Mol Med (2018) 96:469–481 477 Table 2 (continued) Study name Cell source Condition Intervention Primary outcome Study Location Start and References phase end date Conventional treatment and saline was used for the control group Safety study of Human liver-derived Urea cycle HepaStem low dose Safety of HepaStem in Phase I Saint Luc University March 2012– [92] HepaStem for the mesenchymal disorders 12.5 × 10 /kg body paediatric patients and II Hospital, Brussels, April 2015 treatment of urea cycle stem cell Crigler Najjar weight suffering from urea Belgium disorders (UCD) and (HepaStem) syndrome HepaStem intermediate cycle disorder and Universitair Ziekenhuis Crigler-Najjar dose 50 × 10 /kg body Crigler-Najjar (US) Antwerpen, syndrome (CN) weight syndrome Edegem Belgium (HEP001) HepaStem high dose CHU Bicetre, Le Kremlin 200 × 10 /kg body Bic tre Cedex, France weight H pital Jeanne de Flandre, CHRU Lille, Lille Cedex, France H pital des Enfants, CHU de Toulouse, Toulouse, Cedex 9, France Rambam Medical Center, Meyer Children’s Hospital, Haifa, Israel Hadassah Ein-Kerem Medical Center of Israel, Jerusalem, Israel Schneider Children’sMedical Center of Israel, Petach Tikva, Israel Ospedale Pediatrico BambinoGesudiRoma, Roma, Italy Birmingham Children’s Hospital London, London, United Kingdom Macrophage Autologous Liver cirrhosis Autologous activated Liver function Phase I Edinburgh Royal Infirmary August 2016– [93] therapy for liver macrophages macrophages infusion (MELD score) and II Little France Crescent August 2021 cirrhosis via peripheral vein for at 3 months Edinburgh EH16 4SA 30 min. United Kingdom Standard medical care used as the control Information from Clincialtrials.gov [80] 478 J Mol Med (2018) 96:469–481 Patients were monitored over 48 weeks, with the treatment Another promising area could be the use of macrophage group showing an increase in albumin secretion, platelet count therapy to treat liver disease. Macrophages reduce scar tissue and a reduced end-stage liver disease (MELD) score. and stimulate the HPCs to expand and differentiate into mature Furthermore, survival rate after 72 weeks was also higher in hepatocytes. Thomas et al. showed that bone marrow-derived the treatment group compared to the control, with 20.8 and macrophages (BMM) administered to mice with advanced liver 47.4% mortality rate, respectively. The author suggests that fibrosis resulted in a degradation of fibrillar collagen and re- although the mechanism of improved liver function may be duced fibrogenesis. There was also upregulation of the liver unclear, in vivo differentiation of UC-MSC into hepatocytes progenitor cell mitogen tumour necrosis factor-like weak in- is unlikely due to the short period of hepatic recovery and with ducer of apoptosis that was associated with an expansion of only one treatment patient showing increased alpha-fetoprotein the progenitor cell compartment [97]. There are ongoing clini- levels. It is more likely that soluble factors produced by MSCs cal trials to assess the role macrophage therapy could play in may enhance liver revascularization and proliferation [95]. liver cirrhosis [93]. Significant advances have been made to One study has suggested that plasma exchange (PE) helps translate the use of stem cells to promote liver regeneration promote liver regeneration and recovery, leading to UC-MSC and mature hepatic differentiation into clinical use. Currently, differentiation into HLCs. A phase I/II clinical trial is now in most trials are in early phase I/II and results demonstrating the progress, transplanting UC-MSCs into patients with liver fail- efficacy of these techniques are yet to be published. In the near ure. Patients received either conventional treatment (anti-viral future, the full potential of stem cells for liver regeneration in drugs) with UC-MSCs and/or PE treatment, and survival rates patients with liver disease may be better established. were assessed at 48 weeks [84]. For patients with acute-on- chronic liver failure, Promethera Biosciences have developed a product known as HepaStem, which are MSCs that have the Conclusion potential to differentiate into HLCs. A phase IIa clinical trial is now in progress, transplanting these cells via IV injection to HT offers an alternative therapy to OLT with the aim of treating establish the safety and biological efficacy of these cells. liver-based metabolic diseases or ALF. Advances in liver cell Bilirubin, creatinine, INR and albumin values are being therapy are being researched to overcome the obstacles associ- assessed at day 28, 2 months and 1 year post-infusion. In addi- ated with HT, particularly the shortage of healthy donor hepa- tion to using stem cells for liver failure, HLCs are now being tocytes. Although HLCs are promising, no alternative cell used for clinical HT to replace primary hepatocytes in patients source can yet replace the functionality and efficacy of primary with liver-based metabolic disorders. Bone marrow-derived human hepatocytes. MSCs likely hold the greatest attribute as MSCs transdifferentiated into hepatocytes have been immunomodulators, and co-culturing with mature donor hepa- transplanted via the portal vein into patients with familial hy- tocytes. More clinical trials assessing the safety and efficacy of percholesterolemia. Serum cholesterol/LDL levels were HLCs is pivotal before they can be considered as a reliable cell assessed after 6 months to determine the efficacy of the tech- source. In the future, this may allow for liver-based diseases to nique. Furthermore, HepaStem cells are also being used to treat be effectively treated without the need for OLT. patients suffering from urea cycle disorders. Ureagenesis, am- monia values and amino acid levels are being monitored as well Compliance with ethical standards as behaviour, cognitive skills and health-related quality of life indicators for up to 12 months post-infusion [96]. Conflict of interest The authors declare that they have no conflict of MSCs are also being used clinically for immunomodulating interest. therapy in many liver-based applications. One trial is currently Open Access This article is distributed under the terms of the Creative investigating the use of MSCs to promote allograft tolerance Commons Attribution 4.0 International License (http:// and reduce the toxicity that results from exposure to calcineurin creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appro- inhibitors. Paediatric patients receiving a liver transplantation priate credit to the original author(s) and the source, provide a link to the undergo IV injection of bone marrow-derived MSCs. MSC Creative Commons license, and indicate if changes were made. toxicity is being monitored as well as graft function measured by aminotransferase and gamma glutamyl transferase activity, bilirubin, albumin and INR and the individual need for immu- References nosuppressive medication. In addition, MSCs are being used as immunomodulators in ABO-incompatible liver transplantation. 1. 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Journal of Molecular MedicineSpringer Journals

Published: Apr 24, 2018

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