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Cell therapy for advanced liver diseases: Repair or rebuild

Published:September 22, 2020DOI:https://doi.org/10.1016/j.jhep.2020.09.014

      Summary

      Advanced liver disease presents a significant worldwide health and economic burden and accounts for 3.5% of global mortality. When liver disease progresses to organ failure the only effective treatment is liver transplantation, which necessitates lifelong immunosuppression and carries associated risks. Furthermore, the shortage of suitable donor organs means patients may die waiting for a suitable transplant organ. Cell therapies have made their way from animal studies to a small number of early clinical trials. Herein, we review the current state of cell therapies for liver disease and the mechanisms underpinning their actions (to repair liver tissue or rebuild functional parenchyma). We also discuss cellular therapies that are on the clinical horizon and challenges that must be overcome before routine clinical use is a possibility.

      Keywords

      Introduction

      Liver disease represents a significant worldwide health and economic burden, with liver cirrhosis and cancer the 11th and 16th leading causes of death, respectively.
      • Asrani S.K.
      • Devarbhavi H.
      • Eaton J.
      • Kamath P.S.
      Burden of liver diseases in the world.
      The only curative therapy for end-stage liver disease is orthotopic liver transplantation (OLT), but the availability of suitable donor organs falls short of clinical need. In the United States, 11,844 adult and 700 paediatric patients were added to the liver transplant waiting list in 2018, while 8,250 adult and 563 paediatric transplants were performed.
      • Kwong A.
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      • et al.
      OPTN/SRTR 2018 annual data report: liver.
      In Europe, approximately 7,300 liver transplants are performed annually and almost half of patients wait more than 3 months for a transplant.
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      2018 Annual Report of the European Liver Transplant Registry (ELTR) - 50-year evolution of liver transplantation.
      Efforts to overcome this donor shortage have led to surgical grafting techniques, such as heterotopic or partial orthotopic auxiliary transplantation, that provide functional support to enable host liver regeneration.
      • Rela M.
      • Kaliamoorthy I.
      • Reddy M.S.
      Current status of auxiliary partial orthotopic liver transplantation for acute liver failure.
      Split liver transplantation, where 2 grafts are generated from a single donor, can service both an adult and paediatric recipient.
      • Hackl C.
      • Schmidt K.M.
      • Süsal C.
      • Döhler B.
      • Zidek M.
      • Schlitt H.J.
      Split liver transplantation: current developments.
      This is advantageous in paediatric cases, where the grafted liver can grow with the recipient. In 2018, 19.2% of paediatric liver transplants in the US were split liver grafts.
      • Kwong A.
      • Kim W.R.
      • Lake J.R.
      • Smith J.M.
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      • Skeans M.A.
      • et al.
      OPTN/SRTR 2018 annual data report: liver.
      Although current clinical therapies use whole organs or segments to achieve this benefit, therapy at the level of cell transplantation is emerging and may further overcome shortages of organs and reduce the need for invasive surgical procedures.
      There are several theoretical advantages to cell therapy compared to traditional OLT or adjunct grafting. Efficacious hepatocyte transplantation involves reconstitution of as little as 1% of functional tissue for metabolic diseases such as glycogen storage disease 1a,
      • Muraca M.
      • Gerunda G.
      • Neri D.
      • Vilei M.T.
      • Granato A.
      • Feltracco P.
      • et al.
      Hepatocyte transplantation as a treatment for glycogen storage disease type 1a.
      2.5% for acute-on-chronic liver failure (ACLF),
      • Wang F.
      • Zhou L.
      • Ma X.
      • Ma W.
      • Wang C.
      • Lu Y.
      • et al.
      Monitoring of intrasplenic hepatocyte transplantation for acute-on-chronic liver failure: a prospective five-year follow-up study.
      and potentially greater requirements for bridging time to OLT/regeneration in acute liver failure (ALF), raising the possibility of using 1 donor organ for multiple recipients, particularly where cells can be cryopreserved. Cells may be delivered endovascularly which is less invasive than OLT and also preserves the native liver, which, in the context of metabolic disorders, will continue to perform the majority of hepatic functions even if graft failure occurs. Additionally, cell therapy approaches to repair the injury niche may allow host parenchymal regeneration without the need for organ transplant. In this review, we summarise the history of liver cell therapies and the current state-of-the-art.

      Hepatocyte transplant for liver diseases

       Inborn errors of metabolism

      Hepatocyte transplant (HT) is theoretically promising for the treatment of inborn errors of metabolism (IEM), representing a form of ‘cellular gene-therapy’ whereby transplanted hepatocytes containing functional versions of a specific gene can replace host hepatocytes containing dysfunctional disease-causing mutations in said gene. The expansion capacity of serially transplanted hepatocytes in fumarylacetoacetate hydrolase-deficient (Fah−/−) mice, which models hereditary tyrosinemia type-1, demonstrated that adult hepatocytes possess an extraordinary in vivo expansion capacity rivalling that of haematopoietic stem cells.
      • Overturf K.
      • al-Dhalimy M.
      • Ou C.N.
      • Finegold M.
      • Grompe M.
      Serial transplantation reveals the stem-cell-like regenerative potential of adult mouse hepatocytes.
      HT for IEM has demonstrated encouraging short-term clinical results, partially correcting an array of disorders and delaying OLT.
      • Iansante V.
      • Mitry R.R.
      • Filippi C.
      • Fitzpatrick E.
      • Dhawan A.
      Human hepatocyte transplantation for liver disease: current status and future perspectives.
      The first sustained effect of HT for an IEM was the partial correction of hyperbilirubinemia, up to 11 months post-HT, in a 10-year-old patient with Crigler-Najjar syndrome who was infused with 7.5 × 109 hepatocytes via the portal vein.
      • Fox I.J.
      • Chowdhury J.R.
      • Kaufman S.S.
      • Goertzen T.C.
      • Chowdhury N.R.
      • Warkentin P.I.
      • et al.
      Treatment of the Crigler-Najjar syndrome type I with hepatocyte transplantation.
      HT also has demonstrable utility for overcoming donor shortages, by enabling treatment of numerous patients from the same donor or transplantation of marginal tissue. High quality hepatocytes can be isolated and cryopreserved from cadaveric neonatal livers, with greater post-thawing recovery than adult hepatocytes.
      • Tolosa L.
      • Pareja-Ibars E.
      • Donato M.T.
      • Cortés M.
      • López S.
      • Jiménez N.
      • et al.
      Neonatal livers: a source for the isolation of good-performing hepatocytes for cell transplantation.
      Cryopreserved hepatocytes from a 9-day old neonate used to treat children with carbamoyl phosphate synthase 1 deficiency (CPSD; n = 1), citrullinemia (n = 1) or OTC deficiency (n = 2) demonstrated clinical and metabolic stabilisation during initial follow-up for all patients; 1 patient with severe OTC subsequently died from a fatal metabolic decompensation.
      • Meyburg J.
      • Das A.M.
      • Hoerster F.
      • Lindner M.
      • Kriegbaum H.
      • Engelmann G.
      • et al.
      One liver for four children: first clinical series of liver cell transplantation for severe neonatal urea cycle defects.
      Similarly, OTC deficiency was corrected using cryopreserved hepatocytes from remnant liver tissue from a hyper-reduced left lateral segment following a living donor transplantation. The patient was transplanted at 11 days (7.4 × 107 cells) and 14 days of age (6.6 × 107 cells) via the umbilical vein, was discharged 56 days post-HT and was healthy at 3-month follow-up, albeit requiring continued protein restriction, medication for OTC deficiency and immunosuppression.
      • Enosawa S.
      • Horikawa R.
      • Yamamoto A.
      • Sakamoto S.
      • Shigeta T.
      • Nosaka S.
      • et al.
      Hepatocyte transplantation using a living donor reduced graft in a baby with ornithine transcarbamylase deficiency: a novel source of hepatocytes.
      Domino transplant enables the use of tissue from a patient with a metabolic disease that would otherwise be discarded to treat a patient with a different metabolic disease, since only a fraction of functional tissue is required to fulfil function. This approach has demonstrated clinical improvement for a number of disorders including phenylketonuria,
      • Stéphenne X.
      • Debray F.G.
      • Smets F.
      • Jazouli N.
      • Sana G.
      • Tondreau T.
      • et al.
      Hepatocyte transplantation using the domino concept in a child with tetrabiopterin nonresponsive phenylketonuria.
      ,
      • Soltys K.A.
      • Setoyama K.
      • Tafaleng E.N.
      • Soto Gutiérrez A.
      • Fong J.
      • Fukumitsu K.
      • et al.
      Host conditioning and rejection monitoring in hepatocyte transplantation in humans.
      Crigler-Najjar syndrome, propionic acidaemia and CPSD.
      • Celik N.
      • Squires J.E.
      • Soltys K.
      • Vockley J.
      • Shellmer D.A.
      • Chang W.
      • et al.
      Domino liver transplantation for select metabolic disorders: expanding the living donor pool.
      Cell therapy can be used to add cells to the liver, or to remodel and repair the damaged liver.
      In its current form, cell therapy for IEMs is a useful bridge to more permanent therapy. Further work is required to bridge the gap between efficacious pre-clinical HT and sustained clinical success. Theoretical considerations concerning graft function and expansion are outlined herein.
      Transplanted hepatocytes require a competitive proliferative advantage over host cells, as well as appropriate niche signals, to expand and contribute significantly to liver repopulation
      • Grompe M.
      • Laconi E.
      • Shafritz D.A.
      Principles of therapeutic liver repopulation.
      (Fig. 1). For IEMs, this can be observed in animal models of tyrosinemia type 1,
      • Overturf K.
      • al-Dhalimy M.
      • Ou C.N.
      • Finegold M.
      • Grompe M.
      Serial transplantation reveals the stem-cell-like regenerative potential of adult mouse hepatocytes.
      Wilson's disease (Long-Evans cinnamon rat
      • Yoshida Y.
      • Tokusashi Y.
      • Lee G.H.
      • Ogawa K.
      Intrahepatic transplantation of normal hepatocytes prevents Wilson's disease in Long-Evans cinnamon rats.
      ,
      • Park S.M.
      • Vo K.
      • Lallier M.
      • Cloutier A.S.
      • Brochu P.
      • Alvarez F.
      • et al.
      Hepatocyte transplantation in the Long Evans cinnamon rat model of Wilson's disease.
      ) and alpha 1 antitrypsin deficiency (PiZ mice
      • Ding J.
      • Yannam G.R.
      • Roy-Chowdhury N.
      • Hidvegi T.
      • Basma H.
      • Rennard S.I.
      • et al.
      Spontaneous hepatic repopulation in transgenic mice expressing mutant human α1-antitrypsin by wild-type donor hepatocytes.
      ). These disorders affect liver function and architecture, and large-scale repopulation by transplanted hepatocytes is observed. Meanwhile, the negative effects of disorders such as Crigler-Najjar syndrome (Gunn rat
      • Groth C.G.
      • Arborgh B.
      • Björkén C.
      • Sundberg B.
      • Lundgren G.
      Correction of hyperbilirubinemia in the glucuronyltransferase-deficient rat by intraportal hepatocyte transplantation.
      ), hypercholesterolemia (apolipoprotein E knockout mouse
      • Mitchell C.
      • Mignon A.
      • Guidotti J.E.
      • Besnard S.
      • Fabre M.
      • Duverger N.
      • et al.
      Therapeutic liver repopulation in a mouse model of hypercholesterolemia.
      ) and urea cycle disorders (Spf-ash mouse
      • Michel J.L.
      • Rabier D.
      • Rambaud C.
      • Kamoun P.
      • Brousse N.
      • Vassault A.
      • et al.
      ) are largely extrahepatic, and large-scale repopulation and complete functional correction is more difficult into intact parenchyma. Another theoretical consideration is that, in order for functional repopulation to take place, withdrawal from effective therapy (e.g. 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione for tyrosinemia type 1) may be required to give transplanted cells a competitive advantage over host hepatocytes, risking further disease complications.
      Figure thumbnail gr1
      Fig. 1Optimisation of hepatocyte transplantation.
      The first barrier to successful hepatocyte transplant is cell quality, which has been improved by optimising cryopreservation/thawing techniques. Liver preconditioning induces endothelial cell apoptosis, allowing donor cell integration into hepatic cords. The distorted microenvironment of severe injury is challenging for donor engraftment/proliferation. Macrophage TGFβ spreads p21-induced senescence in host and donor hepatocytes. Strategies to mitigate the severe niche include co-transplantation of immunomodulatory cells, inhibiting senescence, or growth factor treatment. Hepatocytes can be encapsulated and transplanted extrahepatically to circumvent the liver injury niche and avoid immune destruction. Successful cell transplant currently provides a functional bridge to host regeneration/orthotopic liver transplantation. ALF, acute liver failure; TGFβ, transforming growth factor-β.

       Acute liver failure

      ALF is a rare condition associated with significant morbidity and mortality that affects patients without pre-existing liver disease.
      • Brennan P.N.
      • Donnelly M.C.
      • Simpson K.J.
      Systematic review: non A-E, seronegative or indeterminate hepatitis; what is this deadly disease?.
      The aetiology of ALF varies geographically, with acetaminophen (APAP) overdose the most common cause of ALF in the UK, USA and parts of Europe,
      • Brennan P.N.
      • Donnelly M.C.
      • Simpson K.J.
      Systematic review: non A-E, seronegative or indeterminate hepatitis; what is this deadly disease?.
      and viral hepatitis most common within Eastern Asia. A significant proportion of ALF cases are idiopathic; so called non A-E, seronegative or indeterminate hepatitis.
      • Brennan P.N.
      • Donnelly M.C.
      • Simpson K.J.
      Systematic review: non A-E, seronegative or indeterminate hepatitis; what is this deadly disease?.
      HT for ALF has met with some clinical success, bridging patients to OLT and, albeit rarely, complete recovery. Some barriers exist for HT to be reliably curative. Transplanted cells encounter a hostile niche for engraftment and expansion with abundant cellular necrosis and apoptosis. Activated macrophages phagocytose cellular debris and initiate compensatory regeneration, but also secrete transforming growth factor-β (TGFβ), which induces hepatocyte replicative senescence, in turn, transmitting senescence to transplanted cells, presenting a barrier to host/donor-mediated regeneration
      • Bird T.G.
      • Müller M.
      • Boulter L.
      • Vincent D.F.
      • Ridgway R.A.
      • Lopez-Guadamillas E.
      • et al.
      TGFβ inhibition restores a regenerative response in acute liver injury by suppressing paracrine senescence.
      (Fig. 1). Pre-clinically, strategies to inhibit paracrine senescence are being developed to overcome this.
      • Bird T.G.
      • Müller M.
      • Boulter L.
      • Vincent D.F.
      • Ridgway R.A.
      • Lopez-Guadamillas E.
      • et al.
      TGFβ inhibition restores a regenerative response in acute liver injury by suppressing paracrine senescence.
      ,
      • Ferreira-Gonzalez S.
      • Lu W.Y.
      • Raven A.
      • Dwyer B.
      • Man T.Y.
      • O'Duibhir E.
      • et al.
      Paracrine cellular senescence exacerbates biliary injury and impairs regeneration.
      Another strategy to circumvent the microenvironment is extrahepatic HT. Sites examined include the peritoneum
      • Nagaki M.
      • Kano T.
      • Muto Y.
      • Yamada T.
      • Ohnishi H.
      • Moriwaki H.
      Effects of intraperitoneal transplantation of microcarrier-attached hepatocytes on D-galactosamine-induced acute liver failure in rats.
      and lymph nodes
      • Hoppo T.
      • Komori J.
      • Manohar R.
      • Stolz D.B.
      • Lagasse E.
      Rescue of lethal hepatic failure by hepatized lymph nodes in mice.
      of mice, with extrahepatic HT enabling graft vascularisation and functional support of injured liver. Alginate microencapsulation of hepatocytes in microbeads protects cells from immune destruction and permits molecular exchange (Fig. 1). The safety of this approach was tested in 8 paediatric patients with ALF. Microbeads were infused intraperitoneally, avoiding the hepatic microenvironment, without immunosuppression. Four patients recovered, avoiding OLT, 3 were bridged to OLT and 1 patient died.
      • Dhawan A.
      • Chaijitraruch N.
      • Fitzpatrick E.
      • Bansal S.
      • Filippi C.
      • Lehec S.C.
      • et al.
      Alginate microencapsulated human hepatocytes for the treatment of acute liver failure in children.
      There are a number of clinical examples of HT for ALF. Relevant cases of acute variants of liver failure treated with HT are shown by indication (Table 1).
      Table 1Hepatocyte transplantation for acute liver failure.
      Number of patientsAge range (years)Survival (death
      Denotes time to death.
      or time to OLT
      Denotes time to OLT.
      )
      Reference
      Drug-induced acute liver failure
       332–556 hours
      Denotes time to death.
      – 20∗ days
      • Bilir B.M.
      • Guinette D.
      • Karrer F.
      • Kumpe D.A.
      • Krysl J.
      • Stephens J.
      • et al.
      Hepatocyte transplantation in acute liver failure.
       421–511 day
      Denotes time to death.
      ->1 year (disease free)
      • Fisher R.A.
      • Strom S.C.
      Human hepatocyte transplantation: worldwide results.
       226,272 days
      Denotes time to OLT.
      –10 days
      Denotes time to OLT.
      • Fisher R.A.
      • Strom S.C.
      Human hepatocyte transplantation: biology and therapy.
       14335 days
      Denotes time to death.
      • Strom S.C.
      • Fisher R.A.
      • Thompson M.T.
      • Sanyal A.J.
      • Cole P.E.
      • Ham J.M.
      • et al.
      Hepatocyte transplantation as a bridge to orthotopic liver transplantation in terminal liver failure.
      Cryptogenic acute liver failure
       1235 days
      Denotes time to OLT.
      –13 days
      Denotes time to death.
      • Sterling R.K.
      • Fisher R.A.
      Liver transplantation. Living donor, hepatocyte, and xenotransplantation.
      Virus-associated acute liver failure
       328–431 day
      Denotes time to OLT.
      –5 days
      Denotes time to death.
      • Strom S.C.
      • Chowdhury J.R.
      • Fox I.J.
      Hepatocyte transplantation for the treatment of human disease.
       1283 days
      Denotes time to OLT.
      • Strom S.C.
      • Fisher R.A.
      • Thompson M.T.
      • Sanyal A.J.
      • Cole P.E.
      • Ham J.M.
      • et al.
      Hepatocyte transplantation as a bridge to orthotopic liver transplantation in terminal liver failure.
       229, 6518 hours
      Denotes time to death.
      , 52 days
      Denotes time to death.
      • Bilir B.M.
      • Guinette D.
      • Karrer F.
      • Kumpe D.A.
      • Krysl J.
      • Stephens J.
      • et al.
      Hepatocyte transplantation in acute liver failure.
       137Fully recovered
      • Fisher R.A.
      • Bu D.
      • Thompson M.
      • Tisnado J.
      • Prasad U.
      • Sterling R.
      • et al.
      Defining hepatocellular chimerism in a liver failure patient bridged with hepatocyte infusion.
       154Day 7
      Denotes time to death.
      • Fisher R.A.
      • Strom S.C.
      Human hepatocyte transplantation: worldwide results.
       14013 hours
      Denotes time to death.
      • Habibullah C.M.
      • Syed I.H.
      • Qamar A.
      • Taher-Uz Z.
      Human fetal hepatocyte transplantation in patients with fulminant hepatic failure.
      Acute-on-chronic liver failure
       719–483 patients fully recovered

      1 survived and subsequently underwent OLT

      3 patients
      Denotes time to death.
      (2.5–12 months)
      • Wang F.
      • Zhou L.
      • Ma X.
      • Ma W.
      • Wang C.
      • Lu Y.
      • et al.
      Monitoring of intrasplenic hepatocyte transplantation for acute-on-chronic liver failure: a prospective five-year follow-up study.
      OLT, orthotopic liver transplantation.
      ∗∗ Denotes time to OLT.
      Denotes time to death.
      HT has been used to treat various drug-induced ALFs, resulting from halothane,
      • Strom S.C.
      • Fisher R.A.
      • Thompson M.T.
      • Sanyal A.J.
      • Cole P.E.
      • Ham J.M.
      • et al.
      Hepatocyte transplantation as a bridge to orthotopic liver transplantation in terminal liver failure.
      dilantin
      • Fisher R.A.
      • Strom S.C.
      Human hepatocyte transplantation: biology and therapy.
      and multiple polysubstance misuse,
      • Fisher R.A.
      • Strom S.C.
      Human hepatocyte transplantation: biology and therapy.
      ,
      • Bilir B.M.
      • Guinette D.
      • Karrer F.
      • Kumpe D.A.
      • Krysl J.
      • Stephens J.
      • et al.
      Hepatocyte transplantation in acute liver failure.
      with improvements in encephalopathy and ammonia concentration. Splenic vein HT successfully bridged 2 adults to OLT at 2 and 10 days post-procedure, respectively,
      • Fisher R.A.
      • Strom S.C.
      Human hepatocyte transplantation: biology and therapy.
      and a 35-year-old adult completely recovered with an isolated intraportal HT of 3.7 × 109 cells. In a single 23-year-old patient with cryptogenic ALF, 2 splenic infusions of donor hepatocytes (2.86 × 109 and 1.52 × 109 cells) bridged the patient to OLT (day 5). However, the patient succumbed to overwhelming sepsis and multiorgan failure at day 13.
      • Sterling R.K.
      • Fisher R.A.
      Liver transplantation. Living donor, hepatocyte, and xenotransplantation.
      Regarding studies of individuals with acute viral pathology and multiorgan dysfunction treated with HT, 2 patients have been successfully bridged to OLT,
      • Strom S.C.
      • Fisher R.A.
      • Thompson M.T.
      • Sanyal A.J.
      • Cole P.E.
      • Ham J.M.
      • et al.
      Hepatocyte transplantation as a bridge to orthotopic liver transplantation in terminal liver failure.
      ,
      • Strom S.C.
      • Chowdhury J.R.
      • Fox I.J.
      Hepatocyte transplantation for the treatment of human disease.
      while a single individual achieved spontaneous recovery following infusion. The majority of these cases were related to acute HBV, with 2 incident cases of acute herpes simplex II virus.
      • Bilir B.M.
      • Guinette D.
      • Karrer F.
      • Kumpe D.A.
      • Krysl J.
      • Stephens J.
      • et al.
      Hepatocyte transplantation in acute liver failure.
      ,
      • Fisher R.A.
      • Strom S.C.
      Human hepatocyte transplantation: worldwide results.
      A variety of delivery routes were used, including intraportal, intrasplenic or intraperitoneal administration. Within this group there was an episode of non-lethal splenic vein thrombosis
      • Sterling R.K.
      • Fisher R.A.
      Liver transplantation. Living donor, hepatocyte, and xenotransplantation.
      and lethal mesenteric vein thrombosis,
      • Fisher R.A.
      • Strom S.C.
      Human hepatocyte transplantation: worldwide results.
      highlighting the risks of such approaches.

       Acute-on-chronic liver failure

      ACLF is characterised by an acute deterioration of liver function in patients with cirrhosis resulting in failure of ≥1 organ(s) and high short-term mortality
      • Jalan R.
      • Gines P.
      • Olson J.C.
      • Mookerjee R.P.
      • Moreau R.
      • Garcia-Tsao G.
      • et al.
      Acute-on chronic liver failure.
      ; ACLF poses unique clinical challenges.
      • Arroyo V.
      • Moreau R.
      • Jalan R.
      • Ginès P.
      • Study E.-C.C.C.
      Acute-on-chronic liver failure: a new syndrome that will re-classify cirrhosis.
      Wang and colleagues trialled intrasplenic HT for ACLF, with a 5-year follow-up interval.
      • Wang F.
      • Zhou L.
      • Ma X.
      • Ma W.
      • Wang C.
      • Lu Y.
      • et al.
      Monitoring of intrasplenic hepatocyte transplantation for acute-on-chronic liver failure: a prospective five-year follow-up study.
      Following transplantation of 4.2 × 1010 to 6.0 × 1010 hepatocytes, 3 patients demonstrated recovery from liver failure, 1 survived but subsequently required OLT, while the remaining 3 patients died 2.5 to 12 months post-HT.

       Summary

      Overall, the literature predominantly comprises individual or small case series with a heterogeneous populous and intrinsic variation in both transplanted cell number and delivery method, making efficacy hard to determine. Future controlled trials with standardised methodology and predetermined and validated endpoints are required to support widespread use of HT. Reassuringly, there appears to be demonstrable longevity in this approach, given that functional hepatocytes were identified at the 48-month interval scan and complete recovery was observed in almost half of patients at 5 years.
      • Wang F.
      • Zhou L.
      • Ma X.
      • Ma W.
      • Wang C.
      • Lu Y.
      • et al.
      Monitoring of intrasplenic hepatocyte transplantation for acute-on-chronic liver failure: a prospective five-year follow-up study.
      Such therapies hold promise as an adjunct to standard of care or as a bridge to OLT. Efforts to improve engraftment and expansion of hepatocytes into intact parenchyma are vital for achieving significant therapeutic efficiency (Fig. 1).

      Overcoming the challenges of cell therapy for liver disease

       Optimising routes of delivery

      Whilst intrasplenic injection is commonly used to access the portal circulation in animal models, the portal vein is typically accessed via 3 routes in humans; ultrasound-guided transcutaneous puncture of an intrahepatic portal vein tributary, intrahepatic transcutaneous splenic vein tributary puncture or via the hepatic venous system with a transjugular intrahepatic portosystemic shunt. These procedures are associated with a bleeding risk,
      • Haddad M.M.
      • Fleming C.J.
      • Thompson S.M.
      • Reisenauer C.J.
      • Parvinian A.
      • Frey G.
      • et al.
      Comparison of bleeding complications between transplenic versus transhepatic access of the portal venous system.
      • Dissegna D.
      • Sponza M.
      • Falleti E.
      • Fabris C.
      • Vit A.
      • Angeli P.
      • et al.
      Morbidity and mortality after transjugular intrahepatic portosystemic shunt placement in patients with cirrhosis.
      • Saad W.E.
      • Madoff D.C.
      Percutaneous portal vein access and transhepatic tract hemostasis.
      compounded by impaired primary haemostasis which can accompany liver failure. Portal vein hypertension frequently accompanies chronic liver disease (CLD), which may progress to the development of portosystemic shunting through varices, reversal of portal vein flow, or portal vein thrombosis, impeding delivery of cells to the liver via this route. The hepatic artery offers an alternate route for cell delivery and is accessed endovascularly, from either common femoral arteries, via the coeliac trunk. Unlike the portal system, retrograde flow and thrombosis do not occur in CLD and the hepatic artery therefore offers an alternate route for cell delivery in cases of portal hypertension. There remains an associated risk of vessel injury which, for the hepatic artery, is dangerous in the context of concurrent portal vein thrombosis. Despite this, the hepatic artery is commonly accessed during transarterial chemoembolisation and selective internal radiation therapy and has been used as a route for cell therapy administration. There is evidence that it may be a more efficient way of delivering cells to the hepatic sinusoid in humans.
      • Khan A.A.
      • Shaik M.V.
      • Parveen N.
      • Rajendraprasad A.
      • Aleem M.A.
      • Habeeb M.A.
      • et al.
      Human fetal liver-derived stem cell transplantation as supportive modality in the management of end-stage decompensated liver cirrhosis.
      Furthermore, the biliary tree is supplied exclusively by the hepatic artery. Therefore, in biliary injury conditions, the hepatic artery offers a more direct delivery route to the portal venous system.
      • Morell C.M.
      • Fabris L.
      • Strazzabosco M.
      Vascular biology of the biliary epithelium.
      Hepatocyte therapy is a paradigm for adding cells to the liver that has been translated to the clinic.

       Improving engraftment and homing

      A significant hurdle to successful cell therapy is the ability of donor cells to access the liver and survive long enough to exert a therapeutic effect. Freshly isolated cells are not readily available, particularly for acute injury where a more “off the shelf” approach is required. Hepatocytes are susceptible to cryopreservation and thawing, with negative effects on survival, engraftment and function, downregulating important adhesion proteins including integrin-β1 and E-cadherin.
      • Terry C.
      • Hughes R.D.
      • Mitry R.R.
      • Lehec S.C.
      • Dhawan A.
      Cryopreservation-induced nonattachment of human hepatocytes: role of adhesion molecules.
      Improvements in parameters such as in vitro apoptosis, in vivo survival, and engraftment (by 4-fold) in APAP-injured mice, were achieved by optimising the cryopreservation medium.
      • Donato M.T.
      • Bolonio M.
      • Cabezas E.
      • Pelechá M.
      • Pareja E.
      • Domènech A.
      • et al.
      Improved in vivo efficacy of clinical-grade cryopreserved human hepatocytes in mice with acute liver failure.
      Thawed hepatocytes display features consistent with early apoptosis. Caspase inhibitors mitigate stress response pathway activation, restoring cell attachment ability and metabolic function.
      • Ölander M.
      • Wiśniewski J.R.
      • Flörkemeier I.
      • Handin N.
      • Urdzik J.
      • Artursson P.
      A simple approach for restoration of differentiation and function in cryopreserved human hepatocytes.
      The use of apoptosis inhibitors is also effective in improving thawed hepatocyte viability and function of alginate-encapsulated and cryopreserved hepatocyte microbeads.
      • Jitraruch S.
      • Dhawan A.
      • Hughes R.D.
      • Filippi C.
      • Lehec S.C.
      • Glover L.
      • et al.
      Cryopreservation of hepatocyte microbeads for clinical transplantation.
      The microenvironment in which cells are transplanted is a critical consideration in the success of cell therapy. Cells enter the liver via sinusoids where they integrate with the parenchyma.
      • Gupta S.
      • Rajvanshi P.
      • Lee C.D.
      Integration of transplanted hepatocytes into host liver plates demonstrated with dipeptidyl peptidase IV-deficient rats.
      • Gupta S.
      • Aragona E.
      • Vemuru R.P.
      • Bhargava K.K.
      • Burk R.D.
      • Chowdhury J.R.
      Permanent engraftment and function of hepatocytes delivered to the liver: implications for gene therapy and liver repopulation.
      • Ponder K.P.
      • Gupta S.
      • Leland F.
      • Darlington G.
      • Finegold M.
      • DeMayo J.
      • et al.
      Mouse hepatocytes migrate to liver parenchyma and function indefinitely after intrasplenic transplantation.
      Specific liver insults, such as portal vein embolisation and partial hepatectomy can pre-condition remaining host hepatocytes to proliferate, positively influencing donor hepatocyte engraftment and proliferation.
      • Michalopoulos G.K.
      Liver regeneration after partial hepatectomy: critical analysis of mechanistic dilemmas.
      ,
      • Gaillard M.
      • Tranchart H.
      • Lainas P.
      • Trassard O.
      • Remy S.
      • Dubart-Kupperschmitt A.
      • et al.
      Improving hepatocyte engraftment following hepatocyte transplantation using repeated reversible portal vein embolization in rats.
      Irradiation-induced apoptosis of sinusoidal endothelial cells allows for greater engraftment and integration of donor hepatocytes into hepatic cords.
      • Yamanouchi K.
      • Zhou H.
      • Roy-Chowdhury N.
      • Macaluso F.
      • Liu L.
      • Yamamoto T.
      • et al.
      Hepatic irradiation augments engraftment of donor cells following hepatocyte transplantation.
      Irradiating individual liver lobes to give donor hepatocytes a competitive advantage has been performed in primates and a patient with phenylketonuria. Biopsy of the patient with phenylketonuria 6 months post-transplant confirmed homing of donor hepatocytes to irradiated areas and Ki67-positive proliferating donor hepatocytes.
      • Soltys K.A.
      • Setoyama K.
      • Tafaleng E.N.
      • Soto Gutiérrez A.
      • Fong J.
      • Fukumitsu K.
      • et al.
      Host conditioning and rejection monitoring in hepatocyte transplantation in humans.
      Other methods to disrupt the sinusoidal endothelial barrier to cellular engraftment include using vasodilating chemicals or agents that induce endothelial injury, such as cyclophosphamide, which increases hepatocyte engraftment in rodents.
      • Malhi H.
      • Annamaneni P.
      • Slehria S.
      • Joseph B.
      • Bhargava K.K.
      • Palestro C.J.
      • et al.
      Cyclophosphamide disrupts hepatic sinusoidal endothelium and improves transplanted cell engraftment in rat liver.
      ,
      • Slehria S.
      • Rajvanshi P.
      • Ito Y.
      • Sokhi R.P.
      • Bhargava K.K.
      • Palestro C.J.
      • et al.
      Hepatic sinusoidal vasodilators improve transplanted cell engraftment and ameliorate microcirculatory perturbations in the liver.
      Influencing the host environment with co-transplantation of cells or factors may also be an effective strategy. HT induces proinflammatory cytokine production by neutrophils and macrophages, reducing hepatocyte engraftment, which can be improved by depleting innate immune cells.
      • Krohn N.
      • Kapoor S.
      • Enami Y.
      • Follenzi A.
      • Bandi S.
      • Joseph B.
      • et al.
      Hepatocyte transplantation-induced liver inflammation is driven by cytokines-chemokines associated with neutrophils and Kupffer cells.
      Co-perfusion with mesenchymal stromal cells (MSCs) could also be beneficial because of their paracrine immunomodulatory potential, as has been demonstrated with islet transplantation. Long-term improvements were made in the engraftment of allogenic pancreatic islet cells into the liver when co-transplanted with GMP-compatible umbilical cord perivascular MSCs, enabling delayed rejection of islet grafts.
      • Forbes S.
      • Bond A.R.
      • Thirlwell K.L.
      • Burgoyne P.
      • Samuel K.
      • Noble J.
      • et al.
      Human umbilical cord perivascular cells improve human pancreatic islet transplant function by increasing vascularization.
      The immunomodulatory function of MSCs alters macrophage phenotype and enhances the pro-regenerative effect of macrophage therapy (discussed later) in pre-clinical murine fibrosis,
      • Watanabe Y.
      • Tsuchiya A.
      • Seino S.
      • Kawata Y.
      • Kojima Y.
      • Ikarashi S.
      • et al.
      Mesenchymal stem cells and induced bone marrow-derived macrophages synergistically improve liver fibrosis in mice.
      and co-transplantation of MSCs improves foetal hepatocyte engraftment in retrorsine-injured mouse livers.
      • Joshi M.
      • B Patil P.
      • He Z.
      • Holgersson J.
      • Olausson M.
      • Sumitran-Holgersson S.
      Fetal liver-derived mesenchymal stromal cells augment engraftment of transplanted hepatocytes.
      Hydrogels have been used to coat cells in substances containing growth factors and matrix proteins to maintain the cells in a pro-regenerative state. A number of materials have been developed including synthetic polyvinyl alcohol
      • Jiang S.
      • Liu S.
      • Feng W.
      PVA hydrogel properties for biomedical application.
      and natural decellularised extracellular matrix (ECM),
      • Lee J.S.
      • Shin J.
      • Park H.M.
      • Kim Y.G.
      • Kim B.G.
      • Oh J.W.
      • et al.
      Liver extracellular matrix providing dual functions of two-dimensional substrate coating and three-dimensional injectable hydrogel platform for liver tissue engineering.
      while coating human epithelial cell adhesion molecule (EpCAM)+ hepatic progenitor cells (HPCs) with hyaluronan increases liver engraftment efficiency by 4.2-fold when delivered via intrasplenic injection
      • Nevi L.
      • Carpino G.
      • Costantini D.
      • Cardinale V.
      • Riccioni O.
      • Di Matteo S.
      • et al.
      Hyaluronan coating improves liver engraftment of transplanted human biliary tree stem/progenitor cells.
      (Fig. 1).

      Alternative sources of hepatocytes

       Foetal liver progenitor cells

      The foetal liver contains EpCAM+/NCAM+ progenitor cells capable of in vitro expansion and in vivo differentiation into hepatocytes
      • Fomin M.E.
      • Beyer A.I.
      • Muench M.O.
      Human fetal liver cultures support multiple cell lineages that can engraft immunodeficient mice.
      ,
      • Schmelzer E.
      • Zhang L.
      • Bruce A.
      • Wauthier E.
      • Ludlow J.
      • Yao H.L.
      • et al.
      Human hepatic stem cells from fetal and postnatal donors.
      (Fig. 2). Epithelial cells isolated from foetal human livers have been transplanted into injured murine livers, promoting fibrosis resolution and functionally regenerating parenchyma.
      • Irudayaswamy A.
      • Muthiah M.
      • Zhou L.
      • Hung H.
      • Jumat N.H.B.
      • Haque J.
      • et al.
      Long-term fate of human fetal liver progenitor cells transplanted in injured mouse livers.
      ,
      • Zhang R.R.
      • Zheng Y.W.
      • Li B.
      • Nie Y.Z.
      • Ueno Y.
      • Tsuchida T.
      • et al.
      Hepatic stem cells with self-renewal and liver repopulation potential are harbored in CDCP1-positive subpopulations of human fetal liver cells.
      Comparisons between the matched repopulation capacity of foetal and adult hepatocytes in immune-deficient liver repopulation mouse models have demonstrated superior repopulation capacity of adult over foetal hepatocytes,
      • Haridass D.
      • Yuan Q.
      • Becker P.D.
      • Cantz T.
      • Iken M.
      • Rothe M.
      • et al.
      Repopulation efficiencies of adult hepatocytes, fetal liver progenitor cells, and embryonic stem cell-derived hepatic cells in albumin-promoter-enhancer urokinase-type plasminogen activator mice.
      ,
      • Hu H.
      • Gehart H.
      • Artegiani B.
      • LÖpez-Iglesias C.
      • Dekkers F.
      • Basak O.
      • et al.
      Long-term expansion of functional mouse and human hepatocytes as 3D organoids.
      which may be a result of the adult liver providing inadequate signals to retain or promote proliferation of immature cell types. Compared to injecting cells in suspension, which lowers engraftment efficacy (as cells migrate to ectopic sites), direct intrahepatic injection of cells within a hyaluronan matrix significantly improves liver engraftment.
      • Turner R.A.
      • Wauthier E.
      • Lozoya O.
      • McClelland R.
      • Bowsher J.E.
      • Barbier C.
      • et al.
      Successful transplantation of human hepatic stem cells with restricted localization to liver using hyaluronan grafts.
      Small case series have been published on human foetal liver cell transplants into patients with CLD,
      • Khan A.A.
      • Shaik M.V.
      • Parveen N.
      • Rajendraprasad A.
      • Aleem M.A.
      • Habeeb M.A.
      • et al.
      Human fetal liver-derived stem cell transplantation as supportive modality in the management of end-stage decompensated liver cirrhosis.
      ,
      • Cardinale V.
      • Carpino G.
      • Gentile R.
      • Napoletano C.
      • Rahimi H.
      • Franchitto A.
      • et al.
      Transplantation of human fetal biliary tree stem/progenitor cells into two patients with advanced liver cirrhosis.
      with reported improvements in biochemistry and clinical findings. In a retrospectively case-controlled study of intrasplenic administration of foetal hepatocytes to 9 patients with CLD, model for end-stage liver disease (MELD) score was stabilised.
      • Pietrosi G.
      • Vizzini G.
      • Gerlach J.
      • Chinnici C.
      • Luca A.
      • Amico G.
      • et al.
      Phases I-II matched case-control study of human fetal liver cell transplantation for treatment of chronic liver disease.
      Further, adequately controlled and powered studies are required to establish efficacy, while problems of inefficient engraftment and expansion must be overcome. Furthermore, ethical controversies surrounding sourcing foetal tissues have limited the clinical adoption of foetal liver tissue transplantation.
      Cell therapies that attempt to remodel and repair the liver and tested in humans include mesenchymal stromal cell and macrophage cell therapy.
      Figure thumbnail gr2
      Fig. 2Hepatocyte donor cell lineages for cell replacement therapy.
      Many different cell types are currently being explored to replace functional parenchymal tissue. Strategies to enable in vitro hepatocyte expansion including 3D organoid expansion, and inducing de-differentiation to revert hepatocytes to CLiPS. Foetal liver progenitors are capable of expansion and clinical transplantation. Expandable HPCs can be antibody-purified from ductal epithelium and regenerate hepatocytes in pre-clinical models of liver disease. Pluripotent cells (ES/iPS cells) can generate hepatocyte-like cells capable of fulfilling hepatocyte functions in vivo, and somatic cells including fibroblasts and MSCs can be reprogrammed to form induced hepatocytes. CLiPS, chemically induced liver progenitor cells; CV, central vein; EpCAM, epithelial cell adhesion molecule; ES, embryonic stem; GSK3, glycogen synthase kinase 3; HA, hepatic artery; HPCs, hepatic progenitor cells; iPS, induced-pluripotent stem; MSCs, mesenchymal stromal cells; NCAM, neural cell adhesion molecule; PV, portal vein; TGFβR, transforming growth factor-β receptor.

       Harnessing innate liver plasticity for cell therapy

      Lineage tracing models of mouse liver injury have established the bi-directional plasticity of liver epithelial cells in rodents.
      • Tarlow B.D.
      • Pelz C.
      • Naugler W.E.
      • Wakefield L.
      • Wilson E.M.
      • Finegold M.J.
      • et al.
      Bipotential adult liver progenitors are derived from chronically injured mature hepatocytes.
      ,
      • Raven A.
      • Lu W.Y.
      • Man T.Y.
      • Ferreira-Gonzalez S.
      • O'Duibhir E.
      • Dwyer B.J.
      • et al.
      Cholangiocytes act as facultative liver stem cells during impaired hepatocyte regeneration.
      When hepatocyte-mediated regeneration is impaired, ductular reactions containing HPCs develop, reconstituting a proportion of hepatocytes (10–15%).
      • Raven A.
      • Lu W.Y.
      • Man T.Y.
      • Ferreira-Gonzalez S.
      • O'Duibhir E.
      • Dwyer B.J.
      • et al.
      Cholangiocytes act as facultative liver stem cells during impaired hepatocyte regeneration.
      ,
      • Deng X.
      • Zhang X.
      • Li W.
      • Feng R.X.
      • Li L.
      • Yi G.R.
      • et al.
      Chronic liver injury induces conversion of biliary epithelial cells into hepatocytes.
      Mouse HPCs can be isolated, expanded and differentiate into hepatocytes when transplanted into pre-clinical models of liver disease. Lgr5+ HPCs are stimulated to proliferate following liver injury and can be isolated and grown as organoids with high clonogenicity. Intrasplenic administration of 0.5 × 106 to 0.8 × 106 HPCs cultured in hepatocyte differentiation medium gave rise to functional parenchymal nodules, up to 1% of the liver, when transplanted into Fah−/− recipients, but was unable to provide functional rescue.
      • Huch M.
      • Dorrell C.
      • Boj S.F.
      • van Es J.H.
      • Li V.S.
      • van de Wetering M.
      • et al.
      In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration.
      Comparable biliary-derived HPCs – harvested and expanded from human livers – can be used to generate small numbers of albumin-producing cells in the liver, when transplanted intrasplenically as undifferentiated HPCs into immune-deficient carbon tetrachloride/retrorsine-injured mice (1–2 × 106 per mouse).
      • Huch M.
      • Gehart H.
      • van Boxtel R.
      • Hamer K.
      • Blokzijl F.
      • Verstegen M.M.
      • et al.
      Long-term culture of genome-stable bipotent stem cells from adult human liver.
      HPCs can be purified with antibodies against ST14 (suppression of tumorigenicity 14),
      • Li B.
      • Dorrell C.
      • Canaday P.S.
      • Pelz C.
      • Haft A.
      • Finegold M.
      • et al.
      Adult mouse liver contains two distinct populations of Cholangiocytes.
      or antibody-based enrichment of non-haematopoietic EpCAM+/CD24+/CD133+ clonally derived progenitors.
      • Lu W.Y.
      • Bird T.G.
      • Boulter L.
      • Tsuchiya A.
      • Cole A.M.
      • Hay T.
      • et al.
      Hepatic progenitor cells of biliary origin with liver repopulation capacity.
      Clonally derived progenitors can be expanded in vitro and transplanted intrasplenically into methionine-choline-deficient mice with induced hepatocyte senescence (imparting a selective advantage for transplanted progenitors; AhCre-Mdm2flox/flox), leading to reconstitution of ~15% of the liver parenchyma from 5 × 106 cells.
      • Lu W.Y.
      • Bird T.G.
      • Boulter L.
      • Tsuchiya A.
      • Cole A.M.
      • Hay T.
      • et al.
      Hepatic progenitor cells of biliary origin with liver repopulation capacity.
      Studies recapitulating hepatocyte de-differentiation to HPC-like cells in vivo, during chronic liver injury, could generate expandable hepatocytes. Using chemical inhibitors of Rho-kinase, TGFβ receptor and glycogen synthase kinase 3, rat hepatocytes are converted into HPC-like cells (chemically induced liver progenitors) that can expand and re-differentiate into functional hepatocyte-like cells, contributing to 72–89% of parenchymal regeneration in urokinase-type plasminogen activator-severe combined immunodeficient mice.
      • Katsuda T.
      • Kawamata M.
      • Hagiwara K.
      • Takahashi R.U.
      • Yamamoto Y.
      • Camargo F.D.
      • et al.
      Conversion of terminally committed hepatocytes to culturable bipotent progenitor cells with regenerative capacity.
      Despite encouraging pre-clinical data, several milestones must be addressed to meet functional and regulatory requirements for clinical translation, including development of good manufacturing practice (GMP)-compliant conditions for culture and cryopreservation, and proving the safety of using stem/progenitor cells for cell therapy. Current methods for organoid growth use clinically incompatible animal tumour-derived matrices.
      • Huch M.
      • Gehart H.
      • van Boxtel R.
      • Hamer K.
      • Blokzijl F.
      • Verstegen M.M.
      • et al.
      Long-term culture of genome-stable bipotent stem cells from adult human liver.
      Efforts to enable clinical translation of liver and other epithelial organoids have explored replacement of undefined culture components with GMP-compatible techniques such as using defined ECM proteins,
      • Jee J.H.
      • Lee D.H.
      • Ko J.
      • Hahn S.
      • Jeong S.Y.
      • Kim H.K.
      • et al.
      Development of collagen-based 3D matrix for gastrointestinal tract-derived organoid culture.
      development of synthetic hydrogels to replace tumour-derived matrices,
      • Broguiere N.
      • Isenmann L.
      • Hirt C.
      • Ringel T.
      • Placzek S.
      • Cavalli E.
      • et al.
      Growth of epithelial organoids in a defined hydrogel.
      and bioengineering scaffolds to recreate organotypic niche interactions.
      • Ng S.S.
      • Saeb-Parsy K.
      • Blackford S.J.I.
      • Segal J.M.
      • Serra M.P.
      • Horcas-Lopez M.
      • et al.
      Human iPS derived progenitors bioengineered into liver organoids using an inverted colloidal crystal poly (ethylene glycol) scaffold.
      De-cellularised liver ECM supports complex in vitro development of foetal liver cells,
      • Vyas D.
      • Baptista P.M.
      • Brovold M.
      • Moran E.
      • Gaston B.
      • Booth C.
      • et al.
      Self-assembled liver organoids recapitulate hepatobiliary organogenesis in vitro.
      and intestinal ECM gels display an overlapping proteomic profile with other endodermal organs, including the liver, and support human adult and foetal organoid expansion, comparable to non-defined matrices.
      • Giobbe G.G.
      • Crowley C.
      • Luni C.
      • Campinoti S.
      • Khedr M.
      • Kretzschmar K.
      • et al.
      Extracellular matrix hydrogel derived from decellularized tissues enables endodermal organoid culture.
      Long-term pre-clinical safety studies are required to ascertain a clinically safe differentiation state of HPCs with no significant tumorigenicity risk. Of particular concern is that biliary cells with constitutively active Wnt and in vivo xenograft tumour-forming ability can be enriched from cirrhotic patients using the biliary lineage marker EpCAM.
      • Khosla R.
      • Rastogi A.
      • Ramakrishna G.
      • Pamecha V.
      • Mukhopadhyay A.
      • Vasudevan M.
      • et al.
      EpCAM+ liver cancer stem-like cells exhibiting autocrine Wnt signaling potentially originate in cirrhotic patients.
      EpCAM-enriched cells from donors with uninjured livers do not appear to contain a tumour-forming component,
      • Khosla R.
      • Rastogi A.
      • Ramakrishna G.
      • Pamecha V.
      • Mukhopadhyay A.
      • Vasudevan M.
      • et al.
      EpCAM+ liver cancer stem-like cells exhibiting autocrine Wnt signaling potentially originate in cirrhotic patients.
      and organoid lines from normal human livers are genetically stable over long-term culture.
      • Huch M.
      • Gehart H.
      • van Boxtel R.
      • Hamer K.
      • Blokzijl F.
      • Verstegen M.M.
      • et al.
      Long-term culture of genome-stable bipotent stem cells from adult human liver.
      These studies provide key proof-of-principle that in vitro expansion and transplantation of HPCs, rather than in vitro-matured progeny, may be a viable strategy for clinical translation with limited tumorigenic risk in the future (Fig. 2). However, the poor engraftment and expansion of immature cells needs to be overcome for progenitor approaches to be clinically viable.

       Pluripotent stem cells

      Hepatocyte-like cells can be derived from pluripotent sources by using factors to recapitulate embryonic development. These factors include Activin A and Wnt3a to induce hepatic endoderm formation,
      • Hay D.C.
      • Fletcher J.
      • Payne C.
      • Terrace J.D.
      • Gallagher R.C.
      • Snoeys J.
      • et al.
      Highly efficient differentiation of hESCs to functional hepatic endoderm requires ActivinA and Wnt3a signaling.
      ,
      • Agarwal S.
      • Holton K.L.
      • Lanza R.
      Efficient differentiation of functional hepatocytes from human embryonic stem cells.
      bone morphogenetic protein and fibroblast growth factor to promote hepatocyte differentiation, and maturation factors hepatocyte growth factor and oncostatin-M.
      • Hay D.C.
      • Zhao D.
      • Fletcher J.
      • Hewitt Z.A.
      • McLean D.
      • Urruticoechea-Uriguen A.
      • et al.
      Efficient differentiation of hepatocytes from human embryonic stem cells exhibiting markers recapitulating liver development in vivo.
      Protocols have been adapted to produce clinical-grade hepatocyte-like cells from embryonic and induced-pluripotent stem cells (iPSCs) that are capable of liver engraftment and function in pre-clinical mouse models.
      • Li Z.
      • Wu J.
      • Wang L.
      • Han W.
      • Yu J.
      • Liu X.
      • et al.
      Generation of qualified clinical-grade functional hepatocytes from human embryonic stem cells in chemically defined conditions.
      ,
      • Blackford S.J.I.
      • Ng S.S.
      • Segal J.M.
      • King A.J.F.
      • Austin A.L.
      • Kent D.
      • et al.
      Validation of current good manufacturing practice compliant human pluripotent stem cell-derived hepatocytes for cell-based therapy.
      However, current protocols are unable to produce fully differentiated hepatocytes from pluripotent cells in vitro and established methods produce cells that are akin to foetal hepatocytes,
      • Baxter M.
      • Withey S.
      • Harrison S.
      • Segeritz C.P.
      • Zhang F.
      • Atkinson-Dell R.
      • et al.
      Phenotypic and functional analyses show stem cell-derived hepatocyte-like cells better mimic fetal rather than adult hepatocytes.
      which is a barrier to clinical utility. This has led to further investigation of different ECM substrates, small molecules, 3D culture systems and assessment of cell of origin for derivation of iPSCs to bridge this gap.
      Culture of human embryonic stem cell-derived hepatocyte-like cells on laminins −521 and −111 suppresses gene expression associated with inhibition of terminal differentiation, enhances CYP1A2 and 3A function and suppresses unwanted fibroblastic and colonic differentiation compared to Matrigel.
      • Cameron K.
      • Tan R.
      • Schmidt-Heck W.
      • Campos G.
      • Lyall M.J.
      • Wang Y.
      • et al.
      Recombinant laminins drive the differentiation and self-organization of hESC-derived hepatocytes.
      Culture of human pluripotent cell-derived hepatocyte-like cells in spheroids under defined culture conditions enables a stable hepatocyte-like phenotype, albeit immature compared to primary hepatocytes, for up to 1 year in vitro; these cells can mitigate the progression of liver injury, but do not rescue function, in FAH-deficient mice.
      • Rashidi H.
      • Luu N.T.
      • Alwahsh S.M.
      • Ginai M.
      • Alhaque S.
      • Dong H.
      • et al.
      3D human liver tissue from pluripotent stem cells displays stable phenotype in vitro and supports compromised liver function in vivo.
      Comparison between isogenic hepatocyte and fibroblast-derived iPSCs determined that the tissue of origin does not affect the functional capacity of hepatocyte progeny,
      • Heslop J.A.
      • Kia R.
      • Pridgeon C.S.
      • Sison-Young R.L.
      • Liloglou T.
      • Elmasry M.
      • et al.
      Donor-dependent and other nondefined factors have greater influence on the hepatic phenotype than the starting cell type in induced pluripotent stem cell derived hepatocyte-like cells.
      and it is donor-dependent genetic variation that affects the hepatocyte-like differentiation capacity of iPSCs.
      • Kajiwara M.
      • Aoi T.
      • Okita K.
      • Takahashi R.
      • Inoue H.
      • Takayama N.
      • et al.
      Donor-dependent variations in hepatic differentiation from human-induced pluripotent stem cells.
      Development of hypoimmunogenic pluripotent cells may overcome immune rejection issues with allogenic transplants.
      • Shani T.
      • Hanna J.H.
      Universally non-immunogenic iPSCs.
      Editing HLA class I and class II molecules,
      • Deuse T.
      • Hu X.
      • Gravina A.
      • Wang D.
      • Tediashvili G.
      • De C.
      • et al.
      Hypoimmunogenic derivatives of induced pluripotent stem cells evade immune rejection in fully immunocompetent allogeneic recipients.
      • Mattapally S.
      • Pawlik K.M.
      • Fast V.G.
      • Zumaquero E.
      • Lund F.E.
      • Randall T.D.
      • et al.
      Human leukocyte antigen class I and II knockout human induced pluripotent stem cell-derived cells: universal donor for cell therapy.
      • Xu H.
      • Wang B.
      • Ono M.
      • Kagita A.
      • Fujii K.
      • Sasakawa N.
      • et al.
      Targeted disruption of HLA genes via CRISPR-Cas9 generates iPSCs with enhanced immune compatibility.
      • Jang Y.
      • Choi J.
      • Park N.
      • Kang J.
      • Kim M.
      • Kim Y.
      • et al.
      Development of immunocompatible pluripotent stem cells via CRISPR-based human leukocyte antigen engineering.
      combined with overexpression of immunoregulatory factors to evade immune recognition, facilitates development of ‘universal donor’ stem cells.
      • Han X.
      • Wang M.
      • Duan S.
      • Franco P.J.
      • Kenty J.H.
      • Hedrick P.
      • et al.
      Generation of hypoimmunogenic human pluripotent stem cells.
      The establishment of hypoimmunogenic universal donor iPSCs that have been developed on a donor genetic background for optimal differentiation to hepatocytes, or non-epithelial cells for niche modulation, would greatly accelerate clinical translation. A limitation to translation is the immaturity of progenitor/stem cell-derived hepatocyte-like cells which preclude significant engraftment and functional rescue. Further studies recapitulating organotypic conditions will aid in the development of fully functional cells for transplant – as well as potentially improving protocols for hepatocyte culture and HPC differentiation – bridging the functional gap between transplanting progenitors vs. mature hepatocytes, and enabling clinical translation (Fig. 2).
      Where the liver is damaged, then techniques to protect the donor cells will be required, such as cell encapsulation.

      Remodelling the injury niche with cell therapy

      An alternative approach to replacing hepatocytes is promoting liver repair by modulating the liver microenvironment and/or systemic immune responses. The hepatic mononuclear phagocytic system and gut co-operate to prevent major immune activation from immunogenic molecules (e.g. bacterial endotoxin) via pattern recognition receptors, and to prevent intestinal bacterial translocation via the portal vein.
      • Jenne C.N.
      • Kubes P.
      Immune surveillance by the liver.
      These cells also mediate the immune response to liver cell death via damage-associated molecular pattern-mediated cytokine/chemokine production and phagocytosis of cellular debris.
      • Dong X.
      • Liu J.
      • Xu Y.
      • Cao H.
      Role of macrophages in experimental liver injury and repair in mice.
      Macrophages play a critical role in this gut-liver axis, representing a second-line defence in sensing invading bacteria or their pathogenic toxins and antigens,
      • Ju C.
      • Tacke F.
      Hepatic macrophages in homeostasis and liver diseases: from pathogenesis to novel therapeutic strategies.
      forming a barrier to peritoneal bacterial infection and sensing bacterial load.
      • Sierro F.
      • Evrard M.
      • Rizzetto S.
      • Melino M.
      • Mitchell A.J.
      • Florido M.
      • et al.
      A liver capsular network of monocyte-derived macrophages restricts hepatic dissemination of intraperitoneal bacteria by neutrophil recruitment.
      Macrophage phagocytic function is diminished in severe injury,
      • Canalese J.
      • Gove C.D.
      • Gimson A.E.
      • Wilkinson S.P.
      • Wardle E.N.
      • Williams R.
      Reticuloendothelial system and hepatocytic function in fulminant hepatic failure.
      compromising their barrier function and leading to infection and innate immune dysfunction, further heightening infection risk.
      • Bernsmeier C.
      • van der Merwe S.
      • Périanin A.
      The innate immune cells in cirrhosis.
      With compromised immunity, bacterial and/or fungal infection is a common occurrence in patients with ALF,
      • Rolando N.
      • Philpott-Howard J.
      • Williams R.
      Bacterial and fungal infection in acute liver failure.
      • Rolando N.
      • Harvey F.
      • Brahm J.
      • Philpott-Howard J.
      • Alexander G.
      • Casewell M.
      • et al.
      Fungal infection: a common, unrecognised complication of acute liver failure.
      • Rolando N.
      • Harvey F.
      • Brahm J.
      • Philpott-Howard J.
      • Alexander G.
      • Gimson A.
      • et al.
      Prospective study of bacterial infection in acute liver failure: an analysis of fifty patients.
      • Wyke R.J.
      • Canalese J.C.
      • Gimson A.E.
      • Williams R.
      Bacteraemia in patients with fulminant hepatic failure.
      cirrhosis
      • Jalan R.
      • Fernandez J.
      • Wiest R.
      • Schnabl B.
      • Moreau R.
      • Angeli P.
      • et al.
      Bacterial infections in cirrhosis: a position statement based on the EASL Special Conference 2013.
      and ACLF,
      • Fernández J.
      • Acevedo J.
      • Wiest R.
      • Gustot T.
      • Amoros A.
      • Deulofeu C.
      • et al.
      Bacterial and fungal infections in acute-on-chronic liver failure: prevalence, characteristics and impact on prognosis.
      ,
      • Mücke M.M.
      • Rumyantseva T.
      • Mücke V.T.
      • Schwarzkopf K.
      • Joshi S.
      • Kempf V.A.J.
      • et al.
      Bacterial infection-triggered acute-on-chronic liver failure is associated with increased mortality.
      driving dysregulated proinflammatory innate immune cell activation, cytokine overactivation and eventually multiorgan failure.
      • Arroyo V.
      • Moreau R.
      • Jalan R.
      • Ginès P.
      • Study E.-C.C.C.
      Acute-on-chronic liver failure: a new syndrome that will re-classify cirrhosis.
      ,
      • Arroyo V.
      • Moreau R.
      • Kamath P.S.
      • Jalan R.
      • Ginès P.
      • Nevens F.
      • et al.
      Acute-on-chronic liver failure in cirrhosis.
      • Yan J.
      • Li S.
      The role of the liver in sepsis.
      • Rolando N.
      • Wade J.
      • Davalos M.
      • Wendon J.
      • Philpott-Howard J.
      • Williams R.
      The systemic inflammatory response syndrome in acute liver failure.
      Cell therapies may have a number of positive functions within the injured liver, by promoting: i) debris removal, ii) scar resolution, iii) epithelial regeneration and iv) host immune cell recruitment. Wider functions may include v) restoring innate immunity and vi) limiting systemic inflammation. Although not applicable to IEMs requiring hepatocyte replacement, ALF, ACLF and cirrhosis (in which uncontrolled injury and inflammation contribute significantly to morbidity and mortality) are particularly amenable to remodelling strategies that reverse the effects of injury. The bone marrow (BM), and its progeny, contains several candidate cell types for this application, including haematopoietic progenitor cells, MSCs and macrophages. The uses of these cell types for treating acute (Fig. 3) and chronic liver injures (Fig. 4) are discussed herein.
      Figure thumbnail gr3
      Fig. 3Cell therapy to modulate the injury niche in acute liver injury.
      DAMP molecules from damaged hepatocytes activate KCs, initiating recruitment of circulating inflammatory monocytes. Macrophages activate γδT-cells via IL-23, which recruit neutrophils. Severe injury impairs macrophage function, allowing gut bacteria translocation, PAMP-induced inflammation, and eventual multiorgan failure. Injury resolution occurs through transition of proinflammatory to pro-resolution macrophages via neutrophil interaction, phagocytosis, anti-inflammatory IL-10 secretion and hepatocyte-mediated regeneration. Exogenous AAMs increase the pro-resolution macrophage pool, inhibit inflammatory monocyte recruitment and restore the innate immune barrier to bacterial translocation. MSCs facilitate pro-resolution phenotypic switching of macrophages and inhibition of inflammation via IL-10. AAMs, alternatively activated macrophages; APAP, acetaminophen; CCL2, C-C motif chemokine ligand 2; CCR2, C-C motif chemokine receptor 2; DAMPs, damage-associated molecular patterns; IL-, interleukin-; KCs, Kupffer cells; MSCs, mesenchymal stromal cells; NLRP3, NLR family pyrin domain containing 3; PAMPs, pathogen-associated molecular patterns.
      Figure thumbnail gr4
      Fig. 4Cell therapy to modulate the injury niche in chronic liver injury.
      Inflammatory macrophages activate hepatic stellate cells to become fibrogenic myofibroblasts via TGFβ and PDGF. Macrophage phenotypic switching drives fibrosis resolution via myofibroblast apoptosis and MMP expression. Recruited pro-resolution immune cells reinforce resolution. Hepatocytes regenerate via proliferation or TWEAK-induced DR. Exogenous HMDMs increase the pro-resolution macrophage pool and recruit host immune cells to reinforce resolution. CAMs recruit host pro-restorative macrophages via CCL2. Immunomodulatory MSCs induce alternatively activated macrophages, amplifying pro-resolution pathways and limit systemic inflammation. HSCs may contribute to macrophages and recruit host immune cells to support resolution. CAMs, classically activated macrophages; CCL2, C-C motif chemokine ligand 2; CCR2, C-C motif chemokine receptor 2; DAMPs, damage-associated molecular patterns; DR, ductular reaction; ECM, extracellular matrix; HMDMs, human monocyte-derived macrophages; HSCs, haematopoietic stem cells; IL-, interleukin-; KCs, Kupffer cells; MMP, matrix metalloprotease; MSCs, mesenchymal stromal cells; PAMPs, pathogen-associated molecular patterns; PDGF, platelet-derived growth factor; TGFβ, transforming growth factor-β.

       Haematopoietic progenitor cells

      Hematopoietic progenitor cells can differentiate into all blood lineages and can be induced to proliferate with granulocyte colony stimulating factor (G-CSF) and enriched using CD34 or CD133 antibodies.
      • Yin A.H.
      • Miraglia S.
      • Zanjani E.D.
      • Almeida-Porada G.
      • Ogawa M.
      • Leary A.G.
      • et al.
      AC133, a novel marker for human hematopoietic stem and progenitor cells.
      ,
      • Civin C.I.
      • Strauss L.C.
      • Brovall C.
      • Fackler M.J.
      • Schwartz J.F.
      • Shaper J.H.
      Antigenic analysis of hematopoiesis. III. A hematopoietic progenitor cell surface antigen defined by a monoclonal antibody raised against KG-1a cells.
      The contribution of BM cells (BMCs) to parenchymal replenishment in liver injury is minor, representing mostly cell fusion events.
      • Thorgeirsson S.S.
      • Grisham J.W.
      Hematopoietic cells as hepatocyte stem cells: a critical review of the evidence.
      • Wang X.
      • Willenbring H.
      • Akkari Y.
      • Torimaru Y.
      • Foster M.
      • Al-Dhalimy M.
      • et al.
      Cell fusion is the principal source of bone-marrow-derived hepatocytes.
      • Vig P.
      • Russo F.P.
      • Edwards R.J.
      • Tadrous P.J.
      • Wright N.A.
      • Thomas H.C.
      • et al.
      The sources of parenchymal regeneration after chronic hepatocellular liver injury in mice.
      Injections of whole BM worsen fibrosis in experimental mouse models
      • Thomas J.A.
      • Pope C.
      • Wojtacha D.
      • Robson A.J.
      • Gordon-Walker T.T.
      • Hartland S.
      • et al.
      Macrophage therapy for murine liver fibrosis recruits host effector cells improving fibrosis, regeneration, and function.
      which may reflect component pro-fibrotic elements such as fibrocytes and MSCs.
      • Kisseleva T.
      • Uchinami H.
      • Feirt N.
      • Quintana-Bustamante O.
      • Segovia J.C.
      • Schwabe R.F.
      • et al.
      Bone marrow-derived fibrocytes participate in pathogenesis of liver fibrosis.
      ,
      • Russo F.P.
      • Alison M.R.
      • Bigger B.W.
      • Amofah E.
      • Florou A.
      • Amin F.
      • et al.
      The bone marrow functionally contributes to liver fibrosis.
      BM also contains matrix metalloprotease (MMP)-producing macrophages that can be antifibrotic.
      • Higashiyama R.
      • Inagaki Y.
      • Hong Y.Y.
      • Kushida M.
      • Nakao S.
      • Niioka M.
      • et al.
      Bone marrow-derived cells express matrix metalloproteinases and contribute to regression of liver fibrosis in mice.
      This complexity is reflected in conflicting pre-clinical and clinical observations when manipulating haematopoietic progenitors in liver disease. G-CSF improves regeneration in murine acute and chronic liver injury,
      • Yannaki E.
      • Athanasiou E.
      • Xagorari A.
      • Constantinou V.
      • Batsis I.
      • Kaloyannidis P.
      • et al.
      G-CSF-primed hematopoietic stem cells or G-CSF per se accelerate recovery and improve survival after liver injury, predominantly by promoting endogenous repair programs.
      and repeat peripheral administration of c-kit+/sca1+/lin- purified BM cells in liver fibrosis reduces collagen deposition in mice.
      • King A.
      • Houlihan D.D.
      • Kavanagh D.
      • Haldar D.
      • Luu N.
      • Owen A.
      • et al.
      Sphingosine-1-phosphate prevents egress of hematopoietic stem cells from liver to reduce fibrosis.
      Interestingly, haematopoietic progenitor cell administration resulted in recruitment of host neutrophils and monocyte-derived macrophages to fibrotic areas. Committed lymphoid progenitors exerted similar antifibrotic effects to haematopoietic progenitors via recruitment of host innate immune cells, suggesting that, rather than exerting a direct antifibrotic effect via differentiation to macrophages, haematopoietic progenitors could modulate host innate immunity to promote resolution of injury
      • King A.
      • Houlihan D.D.
      • Kavanagh D.
      • Haldar D.
      • Luu N.
      • Owen A.
      • et al.
      Sphingosine-1-phosphate prevents egress of hematopoietic stem cells from liver to reduce fibrosis.
      (Fig. 4).
      Uncontrolled and small cohort clinical studies indicate that haematopoietic stem cell therapy can improve liver function,
      • Moore J.K.
      • Stutchfield B.M.
      • Forbes S.J.
      Systematic review: the effects of autologous stem cell therapy for patients with liver disease.
      while larger randomised controlled studies show mixed benefits. A randomised controlled trial reported that G-CSF injection, followed by purification and infusion of autologous haematopoietic stem cells via the portal vein (0.5 × 108 cells; n = 90), stabilised liver biochemistry, improved Child-Pugh score (in 48 cell-treated patients) and increased survival at 6-month follow-up (81/90 alive) compared to placebo water injections (24/50 alive) in patients with HCV-associated end-stage liver disease.
      • Salama H.
      • Zekri A.R.
      • Bahnassy A.A.
      • Medhat E.
      • Halim H.A.
      • Ahmed O.S.
      • et al.
      Autologous CD34+ and CD133+ stem cells transplantation in patients with end stage liver disease.
      However, a multicentre, open-label, phase II trial comparing subcutaneous G-CSF (n = 26), or G-CSF with 3 doses of peripherally infused autologous CD133+ cells (0.2 × 106/kg; n = 28), to standard care (n = 27) in patients with compensated cirrhosis reported no clinical benefit, with worsening of serious adverse events in the treatment arm.
      • Newsome P.N.
      • Fox R.
      • King A.L.
      • Barton D.
      • Than N.N.
      • Moore J.
      • et al.
      Granulocyte colony-stimulating factor and autologous CD133-positive stem-cell therapy in liver cirrhosis (REALISTIC): an open-label, randomised, controlled phase 2 trial.
      These studies differed in terms aetiology (mixed cirrhotic cohort vs. HCV-associated disease), route of delivery (peripheral vs. portal vein infusion), and G-CSF/cell-dosing regimens.

       Mesenchymal stromal cells

      MSCs are multipotent fibroblast-like cells that are characterised by their expression of cell surface antigens CD73, CD90 and CD105, their lack of expression of CD45, CD34, CD14,Cd11b, CD19, CD79α and HLA-DR, their adherence to plastic and their ability to differentiate into osteoclasts, chondrocytes and adipocytes.
      • Dominici M.
      • Le Blanc K.
      • Mueller I.
      • Slaper-Cortenbach I.
      • Marini F.
      • Krause D.
      • et al.
      Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement.
      MSCs were originally identified in BM,
      • Friedenstein A.J.
      • Chailakhjan R.K.
      • Lalykina K.S.
      The development of fibroblast colonies in monolayer cultures of Guinea-pig bone marrow and spleen cells.
      but can be sourced from umbilical and adipose tissue and represent an ideal cell for clinical development as they are easy to isolate, expand and cryopreserve.
      • Bruder S.P.
      • Jaiswal N.
      • Haynesworth S.E.
      Growth kinetics, self-renewal, and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation.
      MSCs exert immunomodulatory effects on T cells, B cells and macrophages, and antifibrotic effects both via immunomodulation and by directly inhibiting hepatic stellate cell proliferation and ECM synthesis.
      • Tsuchiya A.
      • Takeuchi S.
      • Watanabe T.
      • Yoshida T.
      • Nojiri S.
      • Ogawa M.
      • et al.
      Mesenchymal stem cell therapies for liver cirrhosis: MSCs as “conducting cells” for improvement of liver fibrosis and regeneration.
      ,
      • Alfaifi M.
      • Eom Y.W.
      • Newsome P.N.
      • Baik S.K.
      Mesenchymal stromal cell therapy for liver diseases.
      Although some peripherally injected MSCs do reach the liver, cell tracking experiments in humans and rodents have shown that a significant proportion of cells accumulate in the lung and spleen,
      • Watanabe Y.
      • Tsuchiya A.
      • Seino S.
      • Kawata Y.
      • Kojima Y.
      • Ikarashi S.
      • et al.
      Mesenchymal stem cells and induced bone marrow-derived macrophages synergistically improve liver fibrosis in mice.
      ,
      • Gholamrezanezhad A.
      • Mirpour S.
      • Bagheri M.
      • Mohamadnejad M.
      • Alimoghaddam K.
      • Abdolahzadeh L.
      • et al.
      In vivo tracking of 111In-oxine labeled mesenchymal stem cells following infusion in patients with advanced cirrhosis.
      ,
      • Li Q.
      • Zhou X.
      • Shi Y.
      • Li J.
      • Zheng L.
      • Cui L.
      • et al.
      In vivo tracking and comparison of the therapeutic effects of MSCs and HSCs for liver injury.
      and are detectable at lower levels 10 days post-infusion in human cirrhotic patients.
      • Gholamrezanezhad A.
      • Mirpour S.
      • Bagheri M.
      • Mohamadnejad M.
      • Alimoghaddam K.
      • Abdolahzadeh L.
      • et al.
      In vivo tracking of 111In-oxine labeled mesenchymal stem cells following infusion in patients with advanced cirrhosis.
      Even so, transient effects on the immune environment and fibrotic niche may be beneficial in liver disease. The use of MSCs in ALF models attenuates injury via anti-inflammatory interleukin (IL)-10 production, inhibiting hepatocyte death
      • Wang J.
      • Ren H.
      • Yuan X.
      • Ma H.
      • Shi X.
      • Ding Y.
      Interleukin-10 secreted by mesenchymal stem cells attenuates acute liver failure through inhibiting pyroptosis.
      (Fig. 3), and modulates macrophage phenotype in chronic liver injury, increasing the pool of alternatively activated macrophages expressing MMPs to remodel collagen deposits
      • Luo X.Y.
      • Meng X.J.
      • Cao D.C.
      • Wang W.
      • Zhou K.
      • Li L.
      • et al.
      Transplantation of bone marrow mesenchymal stromal cells attenuates liver fibrosis in mice by regulating macrophage subtypes.
      (Fig. 4).
      The gap from animal models to clinical testing of cell therapies needs to be lessened. Ways to do this may include the use of clinically realistic animal models and the development of predictive mathematical models.
      Clinical trials utilising MSCs have been completed for a variety of liver indications with mixed results. In a randomised placebo-controlled trial, peripherally infused autologous MSCs had no beneficial effect on biochemical parameters of cirrhosis in patients with decompensated cirrhosis, with 3 out of 15 patients in the cell treatment group dying in the first 5 months of the study compared to none of the control patients (n = 12).
      • Mohamadnejad M.
      • Alimoghaddam K.
      • Bagheri M.
      • Ashrafi M.
      • Abdollahzadeh L.
      • Akhlaghpoor S.
      • et al.
      Randomized placebo-controlled trial of mesenchymal stem cell transplantation in decompensated cirrhosis.
      Where some efficacy is observed, the effects of MSCs are generally transient. In patients with post-HCV end-stage liver disease, MSC-treated patients showed stabilisation of biochemical measures of liver function compared to worsening in control patients (n = 20 per group), but no significant difference in markers of collagen remodelling.
      • Salama H.
      • Zekri A.R.
      • Medhat E.
      • Al Alim S.A.
      • Ahmed O.S.
      • Bahnassy A.A.
      • et al.
      Peripheral vein infusion of autologous mesenchymal stem cells in Egyptian HCV-positive patients with end-stage liver disease.
      A randomised, controlled study of cirrhotic patients assessed 1 or 2 cycles of portal vein infusion of haematopoietic stem cells followed by peripheral infusion of 1 × 106/kg differentiated BM-MSCs 8 days after haematopoietic stem cell infusion (n = 30 per group), showing an average MELD reduction of 3.27 in doubly infused patients compared with a 0.88 reduction in controls. Improvements in Child-Pugh grade and liver biochemistry were also observed with MSC therapy. Importantly, HCC incidence was not altered with MSC therapy.
      • Zekri A.R.
      • Salama H.
      • Medhat E.
      • Musa S.
      • Abdel-Haleem H.
      • Ahmed O.S.
      • et al.
      The impact of repeated autologous infusion of haematopoietic stem cells in patients with liver insufficiency.
      Likewise, a study assessing autologous BM-MSC therapy via the hepatic artery in 53 patients with chronic HBV-related liver failure vs. 105 injury-matched controls showed improvements in MELD, bilirubin, albumin and prothrombin time in both treated and control groups, with significant improvements in MELD in treatment vs. control groups and no change in HCC incidence or survival at 192 weeks.
      • Peng L.
      • Xie D.Y.
      • Lin B.L.
      • Liu J.
      • Zhu H.P.
      • Xie C.
      • et al.
      Autologous bone marrow mesenchymal stem cell transplantation in liver failure patients caused by hepatitis B: short-term and long-term outcomes.
      A phase II randomised open-label study in 72 patients with alcohol-related cirrhosis assessed 1 or 2 infusions of 50 million BM-MSCs delivered via the hepatic artery. The authors reported a 25% and 37% reduction in collagen area in liver biopsies from pre- to 6 months post single or double BM-MSC infusion, respectively, with improvements in Child-Pugh score, but not MELD, with cell treatment, and no difference in adverse events between groups.
      • Suk K.T.
      • Yoon J.H.
      • Kim M.Y.
      • Kim C.W.
      • Kim J.K.
      • Park H.
      • et al.
      Transplantation with autologous bone marrow-derived mesenchymal stem cells for alcoholic cirrhosis: phase 2 trial.
      In an open-label, non-blinded randomised control trial of 56 patients receiving repeat infusions of allogenic BM-MSCs, treatment improved survival (73.2% vs. 55.6%, p = 0.03) and reduced the incidence of severe infections in hepatitis B-associated ACLF. Cell infusion was associated with fever in these patients,
      • Lin B.L.
      • Chen J.F.
      • Qiu W.H.
      • Wang K.W.
      • Xie D.Y.
      • Chen X.Y.
      • et al.
      Allogeneic bone marrow-derived mesenchymal stromal cells for hepatitis B virus-related acute-on-chronic liver failure: a randomized controlled trial.
      but despite this side effect this data suggests the immune modulatory function of MSCs could potentially be beneficial in ACLF.
      MSCs can be partially differentiated into hepatocyte-like cells with some in vitro function, including CYP3A4 activity and urea production. But, these MSC-derived cells retain CD90 expression, express less albumin and do not upregulate HNF4α or CYP2B6,
      • Campard D.
      • Lysy P.A.
      • Najimi M.
      • Sokal E.M.
      Native umbilical cord matrix stem cells express hepatic markers and differentiate into hepatocyte-like cells.
      providing evidence of a lack of complete transdifferentiation (Fig. 2). Pre-clinically, they can support some metabolic functions and liver repopulation, but still retain mesenchymal marker expression
      • Najimi M.
      • Khuu D.N.
      • Lysy P.A.
      • Jazouli N.
      • Abarca J.
      • Sempoux C.
      • et al.
      Adult-derived human liver mesenchymal-like cells as a potential progenitor reservoir of hepatocytes?.
      and have been assessed in a phase I safety trial in paediatric metabolic liver disorders, although without comparison to undifferentiated MSCs.
      • Smets F.
      • Dobbelaere D.
      • McKiernan P.
      • Dionisi-Vici C.
      • Broué P.
      • Jacquemin E.
      • et al.
      Phase I/II trial of liver-derived mesenchymal stem cells in Pediatric liver-based metabolic disorders: a prospective, open label, multicenter, partially randomized, safety study of one cycle of heterologous human adult liver-derived progenitor cells (HepaStem) in urea cycle disorders and Crigler-Najjar syndrome patients.
      Conversely, a phase II non-randomised study comparing undifferentiated MSCs (n = 9) to hepatocyte-like MSCs (n = 6), or standard care control (n = 10) showed short-term improvements in MELD, bilirubin, albumin and clinical signs of liver disease, in patients receiving MSC therapy for HCV. No difference in clinical effect was observed between patients receiving undifferentiated MSCs or hepatocyte-differentiated MSCs,
      • El-Ansary M.
      • Abdel-Aziz I.
      • Mogawer S.
      • Abdel-Hamid S.
      • Hammam O.
      • Teaema S.
      • et al.
      Phase II trial: undifferentiated versus differentiated autologous mesenchymal stem cells transplantation in Egyptian patients with HCV induced liver cirrhosis.
      suggesting that the contribution to hepatocyte functions from MSCs is negligible. Reprogramming somatic cells, such as fibroblasts or MSCs, with hepatic transcription factors to produce induced hepatocytes (iHeps) is also being explored.
      • Du Y.
      • Wang J.
      • Jia J.
      • Song N.
      • Xiang C.
      • Xu J.
      • et al.
      Human hepatocytes with drug metabolic function induced from fibroblasts by lineage reprogramming.
      ,
      • Huang P.
      • Zhang L.
      • Gao Y.
      • He Z.
      • Yao D.
      • Wu Z.
      • et al.
      Direct reprogramming of human fibroblasts to functional and expandable hepatocytes.
      iHeps display a transcriptomic profile and functionality approaching that of human hepatocytes and significantly repopulate Fah−/− livers, improving 27-day survival from 0% to 40%.
      • Sekiya S.
      • Suzuki A.
      Direct conversion of mouse fibroblasts to hepatocyte-like cells by defined factors.
      Long-term expanded MSC-derived iHeps undergo uncontrolled proliferation and a reversion to a mixed epithelial/adipocyte phenotype, with adverse outcomes when transplanted into a model of hepatocellular injury and senescence – the AhCre-Mdm2flox/flox mouse
      • Orge I.D.
      • Gadd V.L.
      • Barouh J.L.
      • Rossi E.A.
      • Carvalho R.H.
      • Smith I.
      • et al.
      Phenotype instability of hepatocyte-like cells produced by direct reprogramming of mesenchymal stromal cells.
      – raising concerns regarding the phenotypic stability of iHeps when transplanted into an adverse hepatic niche.

       Macrophages

      Recruited monocyte-derived macrophages play a dual role in orchestrating the proinflammatory response to liver injury, which mediates the recruitment of immune cells to the injury niche, activation of hepatic stellate cells to promote liver fibrosis
      • Wilhelm A.
      • Shepherd E.L.
      • Amatucci A.
      • Munir M.
      • Reynolds G.
      • Humphreys E.
      • et al.
      Interaction of TWEAK with Fn14 leads to the progression of fibrotic liver disease by directly modulating hepatic stellate cell proliferation.
      ,
      • Ramachandran P.
      • Dobie R.
      • Wilson-Kanamori J.R.
      • Dora E.F.
      • Henderson B.E.P.
      • Luu N.T.
      • et al.
      Resolving the fibrotic niche of human liver cirrhosis at single-cell level.
      and initiation of progenitor-mediated liver regeneration and hepatocytic differentiation.
      • Boulter L.
      • Govaere O.
      • Bird T.G.
      • Radulescu S.
      • Ramachandran P.
      • Pellicoro A.
      • et al.
      Macrophage-derived Wnt opposes Notch signaling to specify hepatic progenitor cell fate in chronic liver disease.
      ,
      • Tirnitz-Parker J.E.
      • Viebahn C.S.
      • Jakubowski A.
      • Klopcic B.R.
      • Olynyk J.K.
      • Yeoh G.C.
      • et al.
      Tumor necrosis factor-like weak inducer of apoptosis is a mitogen for liver progenitor cells.
      In ALF, administration of CSF-1 (colony stimulating factor 1) promotes differentiation of infiltrating monocytes, restoration of liver innate immunity and accelerated recovery in a mouse model of APAP-induced injury.
      • Stutchfield B.M.
      • Antoine D.J.
      • Mackinnon A.C.
      • Gow D.J.
      • Bain C.C.
      • Hawley C.A.
      • et al.
      CSF1 restores innate immunity after liver injury in mice and serum levels indicate outcomes of patients with acute liver failure.
      Phagocytosis of debris by macrophages and interaction with neutrophils mediates a phenotypic shift from proinflammatory (‘M1-like’) to pro-resolution (‘M2-like’) macrophages via downregulation of the proinflammatory sensor NLRP3 (NLR family pyrin domain containing 3) and upregulation of anti-inflammatory IL-10, which reinforces a pro-phagocytic phenotype by downstream STAT3 signalling and autocrine IL-6 stimulation.
      • Campana L.
      • Starkey Lewis P.J.
      • Pellicoro A.
      • Aucott R.L.
      • Man J.
      • O'Duibhir E.
      • et al.
      The STAT3-IL-10-IL-6 pathway is a novel regulator of macrophage efferocytosis and phenotypic conversion in sterile liver injury.
      • Calvente C.J.
      • Tameda M.
      • Johnson C.D.
      • Del Pilar H.
      • Lin Y.C.
      • Adronikou N.
      • et al.
      Neutrophils contribute to spontaneous resolution of liver inflammation and fibrosis via microRNA-223.
      • Ramachandran P.
      • Pellicoro A.
      • Vernon M.A.
      • Boulter L.
      • Aucott R.L.
      • Ali A.
      • et al.
      Differential Ly-6C expression identifies the recruited macrophage phenotype, which orchestrates the regression of murine liver fibrosis.
      Pro-resolution macrophages upregulate pro-phagocytic genes and collagen-degrading MMPs.
      • Ramachandran P.
      • Pellicoro A.
      • Vernon M.A.
      • Boulter L.
      • Aucott R.L.
      • Ali A.
      • et al.
      Differential Ly-6C expression identifies the recruited macrophage phenotype, which orchestrates the regression of murine liver fibrosis.
      Mer tyrosine kinase (MerTK) is a phagocytic receptor that recognises apoptotic cells, facilitating the functional switch from proinflammatory to pro-resolution macrophages following efferocytosis of cargo
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      These data provide a fundamental link between liver inflammation, regeneration, wound healing and resolution processes orchestrated by macrophages, highlighting the critical role interactions between neutrophils and macrophages play in the switch from a proinflammatory to a pro-resolution phenotype (with immunomodulatory and injury resolution properties) (Fig. 3).
      Given that recruited macrophages play a crucial role in orchestrating liver repair and regeneration, exogenous ex vivo-differentiated macrophages with the appropriate phenotype could be administered to accelerate liver disease regression. In acute liver injury, intravenous administration of either mouse or clinical-grade human IL4/IL13-treated alternatively activated macrophages, with enhanced phagocytic capacity over naïve macrophages, reduced liver necrotic area, reduced liver infiltration of Ly6Chi inflammatory monocytes and reduced proinflammatory cytokines in serum and liver
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      (Fig. 3). In chronic disease, M-CSF differentiated BM-derived macrophages administered via venous or intrasplenic injection reduced liver scar formation via the production of MMPs, increased the regenerative ductular reaction and recruited host immune cells, such as neutrophils and monocytes, to the injury niche to reinforce injury repair mechanisms
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      (Fig. 4). Macrophages administered via the circulation home to both the liver and spleen in uninjured and APAP-injured mice.
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      suggesting minimal Kupffer cell reconstitution by donor cells in pre-clinical models. However, recruitment of host innate immune cells is a critical mechanism for macrophage therapy and can be enhanced by polarising macrophages to a classically activated macrophage (CAM) phenotype with lipopolysaccharide and interferon-γ.
      • Ma P.F.
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      Role of macrophages in experimental liver injury and repair in mice.
      CAMs upregulate the monocyte chemoattractant CCL2 (C-C motif chemokine ligand 2), which increases recruitment of host monocytes to the injury niche, where they become Ly6Clow restorative macrophages, expressing key phagocytosis genes such as MerTK, MARCO and TREM2,
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      analogous to post phagocytic pro-resolution macrophages,
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      • et al.
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      (Fig. 4). Clinically, a dose escalation trial of autologous macrophage therapy for cirrhosis demonstrated safety in 9 patients with compensated cirrhosis, up to a maximum assessed dose of 109 cells per patient of M-CSF-differentiated monocyte-derived macrophages delivered via peripheral vein injection,
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      with efficacy currently being assessed in an ongoing phase II randomised controlled trial (ISRCTN 10368050).

      Conclusions

      There has been encouraging progress in the development of cell replacement methods to reconstitute liver parenchyma, including hepatocyte cryopreservation and thawing, cell encapsulation to prevent immune rejection, and host preconditioning studies to improve engraftment and survival. Much progress has been made in developing stem/progenitor sources of hepatocytes. However, the development of GMP-compatible cell production methods is required for clinical safety and efficacy testing. Alternative remodelling strategies that act directly to reverse liver injury dynamics, and harness host innate immunity to reignite stalled resolution processes, are being assessed in clinical trials with encouraging results, but much more work is required to rigorously establish efficacy. For cell types which are not as far advanced along the clinical pipeline, particularly stem cell-derived therapies, barriers to be overcome include proving long-term safety and improving engraftment and maturity of cells to enhance efficacy. Nonetheless, with approaches to repair or rebuild the injured liver being assessed in patients, cell therapy for liver disease is on the cusp of meaningful clinical utility.

       Abbreviations

      ACLF, acute-on-chronic liver failure; ALF, acute liver failure; APAP, acetaminophen; BM, bone marrow; BMC, bone marrow cell; CAM, classically activated macrophage; CLD, chronic liver disease; ECM, extracellular matrix; EpCAM, epithelial cell adhesion molecule; FAH, fumarylacetoacetate hydrolase; G-CSF, granulocyte colony stimulating factor; HPC, hepatic progenitor cells; HT, hepatocyte transplant; IEM, inborn errors of metabolism; iHeps, induced hepatocytes; IL-, interleukin-; MELD, model for end-stage liver disease; MerTK, Mer tyrosine kinase; MMP, matrix metalloprotease; MSC, mesenchymal stromal cells; NCAM, neural cell adhesion molecule; OLT, orthotopic liver transplantation; TGFβ, transforming growth factor-β.

      Financial support

      M.T.M. is supported by funds from Wellcome Trust . S.J.F. is supported by funds from Wellcome Trust , Medical Research Council , UKRMP and Syncona Ltd .

      Authors' contributions

      B.J.D., M.T.M, P.N.B and S.J.F. contributed to writing the original draft and review and editing of the final manuscript.

      Conflict of interest

      S.J.F. has patents pending, entitled ‘Macrophage-based therapy’ in national territories of USA, Europe, Japan, China and Australia. These patents have been derived from PCT/GB2017/052769 filed 18/09/2017 and claim priority from UK application 1615923.8 filed 19/09/2016. Both of the original patents have now been abandoned because the original UK patent and PCT patent are no longer live and have now been replaced by the national patents. B.J.D. and S.J.F have patents pending regarding macrophage cell therapy, UK applications 2007903.4, 2007906.7 and 2007905.9, filed on 27/05/2020.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Supplementary data

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