Clinical Hepatocyte Transplantation: What Is Next?

Abstract

Purpose of review

Significant recent scientific developments have occurred in the field of liver repopulation and regeneration. While techniques to facilitate liver repopulation with donor hepatocytes and different cell sources have been studied extensively in the laboratory, in recent years clinical hepatocyte transplantation (HT) and liver repopulation trials have demonstrated new disease indications and also immunological challenges that will require the incorporation of a fresh look and new experimental approaches.

Recent findings

Growth advantage and regenerative stimulus are necessary to allow donor hepatocytes to proliferate. Current research efforts focus on mechanisms of donor hepatocyte expansion in response to liver injury/preconditioning. Moreover, latest clinical evidence shows that important obstacles to HT include optimizing engraftment and limited duration of effectiveness, with hepatocytes being lost to immunological rejection. We will discuss alternatives for cellular rejection monitoring, as well as new modalities to follow cellular graft function and near-to-clinical cell sources.

Summary

HT partially corrects genetic disorders for a limited period of time and has been associated with reversal of ALF. The main identified obstacles that remain to make HT a curative approach include improving engraftment rates, and methods for monitoring cellular graft function and rejection. This review aims to discuss current state-of-the-art in clinical HT and provide insights into innovative approaches taken to overcome these obstacles.

Introduction

Hepatocyte transplantation (HT) has for some time been seen as a promising potential alternative to orthotopic liver transplantation (OLT) expanding the therapeutic approach to a collection of liver diseases. Advantages of cell transplantation compared to whole-organ replacement include a less invasive procedure, the ability of multiple recipients to benefit from a single donor, the capacity to cryopreserve cells for long-term storage and use at later time points, improved theoretical safety profile as graft rejection reverts the patient to their pretransplant state, and the inherent preservation of the native liver so as to enable potential recovery in cases such as acute liver failure from acetaminophen induced liver injury (1). In general, ideal candidate conditions for HT therapy include those where the hepatic scaffolding and microenvironment are preserved and vasculature remains intact. Certain monogenic diseases of the liver exemplify such conditions where there is primary hepatic expression of single gene defects without significant parenchymal damage. In such instances, HT is suggested to enable the replacement of a critical mass of metabolically normal cells with a functioning gene to support appropriate metabolic processes. Acute liver failure (ALF) represents another opportunity for HT therapy. Here, HT aims to temporarily improve liver function and allow the natural regenerative capacity of the liver to proceed or to stabilize and bridge affected patients to more traditional liver transplant. Indeed, published experiences of HT in the treatment of liver-based metabolic diseases (2–16) and ALF (17–21) have shown promising early results; however, only partial correction of metabolic disorders has been achieved and HT has not shown to reliably circumvent the need for traditional organ transplant in ALF (5, 6, 8, 12, 14, 21–25). Thus, in many ways HT has yet to live up to its expectations. This is underscored by the declining number of active hepatocyte transplantation programs (26, 27).

Identifying an ample source of hepatocytes for transplant, optimizing cell quality and storage, maximizing engraftment, controlling rejection episodes, and the ability to monitor the function and rejection of transplanted cells in vivo constitute the main challenges preventing broader implementation of HT into clinical treatment algorithms.

Hepatocyte cell transplantation involves supplementing approximately 5–10% of the native liver volume with human hepatocytes harvested from donor livers (28, 29). HT has been performed in many clinical conditions with varying success. (Table 1) A recent review of the clinical outcomes of the first 100 patients treated with HT has been published (30) and an additional 43 patient experiences have been reported (26). While a detailed clinical report is beyond the scope of this article, it is noted that significant barriers persist limiting broader HT implementation (26, 31, 32) explicitly highlighted in a recent report that included a preclinical and clinical approach (25).The identification of these impediments has led to recent advancements looking to overcome the limitations.

Table 1.

Experience of hepatocyte transplantation in human diseases

Inborn errors of metabolism	Acute liver failure
Acute intermittent porphyria	Acute fatty liver of pregnancy
a1-Antitrypsin deficiency	Drug induced
Crigler-Najjar syndrome	Idiopathic
Factor VII deficiency	Mushroom poisoning
Familial hypercholesterolemia	Post-surgical
Glycogen storage diseases	Viral
Hemochromatosis-hemosiderosis
Hyperlipidemia	Other
Infantile Refsum's disease	Biliary atresia
Primary oxalosis	Cirrhosis
Phenylketonuria
PFIC 2, ABCB11 deficiency, Bile salt exporter pump disease
Tyrosinemia
Urea cycle defects
Ornithine transcarba mylase
deficiency
Argininosuccinate lyase deficiency
Carbamoylphosphate synthase type 1 deficiency
Citrullinemia

PFIC, Progressive familial intrahepatic cholestasis

The aim of this report is to critically evaluate the current state of HT, underscoring important advancements that have occurred in the expanding field of liver cell transplantation and discuss new exciting technologies that have the potential to change the landscape of liver replacement once and for all.

Improving Human Hepatocytes Quality

A growing interest in cell therapies has enabled multiple avenues of research investigating the benefits in treating a variety of liver diseases. The use of fully differentiated human hepatocytes is preferred to alternative cell sources, such as stem cells and their derivatives, due to minimizing the concern for cancerous transformation and their poor ability to function as primary human hepatocytes (33–36). Currently, the primary sources of hepatocytes for HT remains livers that have been denied for OLT, surplus tissue resulting from reduce-graft transplants, and normal tissue resulting from benign tumor resection procedures (37–39). Recent efforts looking to expand the sources of human hepatocytes have shown that explanted diseased livers might represent a valuable source of metabolically competent human hepatocytes (40). Regardless of the source, other small studies have shown that a variety of factors affect the yield and viability of the hepatocytes isolated from these donor livers, sometimes with conflicting results, and multiple factors are often present within a single donor specimen (39, 41–48). These findings, combined with the knowledge that the quality of isolated hepatocytes is directly correlated with clinical outcomes after HT, has led investigators to search for strategies to optimize the cell product in hepatocyte isolation. A recent investigation, utilizing over 1000 samples, has greatly expanded the understanding of the interplay among various factors affecting viability and yield of isolated human hepatocytes (49). (Table 2) The investigators then used the data to generate algorithms for the prediction of isolated hepatocyte viability and yield which have been made publicly available (www.klinikum.uni-uenchen.de/Chirurgische-Klinik-und-Poliklinik-Grosshadern/de/0700-forschung/ag-leberregeneration/core-facility/Qualitaetsrechner_Hepatozyten.html). While predictive modeling is provocative, ultimate hepatocyte isolation success is determined by validated methodology and protocols. Therefore, modifications aimed at improving the quality of isolated human hepatocytes are constantly being explored.

Table 2.

Factors affecting yield and viability of isolated hepatocytes

	Yield	Viability
Fibrosis	↓	↓
Chemotherapy	↑
Steatosis	↓	↓
Liver enzyme and bilirubin elevation	↓	↓
Weight of perfused liver	↓
Cold ischemia time	↓

Ref 49

Recent introduction of liberase, an enzyme used in pancreatic islet cell isolation, and N-acetylcysteine (NAC), an antioxidant with multiple hepatoprotective properties, into established isolation protocols has enabled improvements in overall isolation success from 40% to 70% (33, 50, 51). Additional strategies such as machine perfusion techniques and perfusate selection have been shown to dramatically increase cell yields (52, 53). Moreover, machine perfusion organ reconditioning has proven valuable in improving cell viability and function of ischemic livers and experimentally on fatty livers (53–56). Alternatively, the addition of polyethylene glycol to hypothermic preservation solutions has been shown to improve hepatocyte survival following successful isolation (57–59). Ultimately, definitive assessment of hepatocyte quality is critical prior to proceeding with HT. Novel approaches with the capacity to quickly and affordably assess a variety of hepatic functions, as a reflection of quality, are enabling more personalization of HT preparations (26, 60, 61). Future work aimed at the standardization of the procedural techniques ranging from isolation through cryopreservation, thawing, and functional assessment of hepatocytes prior to HT will enable the incorporation of various center experiences into longitudinal studies advancing the field of HT in humans.

Improving Transplanted Hepatocytes Engraftment and Repopulation

After decades of experimental and clinical trials, we now know that 1) a selective growth advantage over native hepatocytes and 2) regenerative stimuli are necessary to allow donor hepatocytes to proliferate after hepatocyte transplantation. Engraftment describes the process by which transplanted hepatocytes translocate from the sinusoidal space into the recipient liver plates following disruption of the sinusoidal endothelium and integrate into the host liver parenchyma. This process happens soon after the vascular delivery of hepatocytes into the native liver and can last for a few days, but remains relatively inefficient with potential loss of >70% of infused cells (62). Once hepatocytes have engrafted in the liver sinusoids, transplanted hepatocytes should be induced to repopulate the liver parenchymal by providing growth advantage and a regenerative stimulus. Published reports describe a range of variability (6, 11, 15, 26) and efforts to provide donor cells a selective growth advantage over the host liver include partial hepatectomy (26, 63) (short-term regenerative stimulus), preoperative portal vein occlusion (32, 64, 65) (short-term regenerative stimulus), and native liver irradiation (26, 66–69) (growth advantage). However, it is acknowledged that some of these techniques, such as major hepatectomy and chemotherapeutic irradiation, carry unacceptably high risks for human patients limiting their use (13, 32, 33, 68, 69). Since the mechanism of these approaches are essentially different; providing either regeneration stimulus or growth advantage, clinical approaches that seek to deliver both are desirable to maximize the potential of donor hepatocytes to repopulate livers.

Segmental portal embolization, by causing transitional ischemia, provides a strong regenerative stimulus and has demonstrated good regeneration capacity with acceptable risk (13, 70). In this procedure, a balloon is positioned in the left portal vein just beyond the bifurcation. It is inflated so as to occlude the left portal vein but allows transportal infusion of cells into the right lobe of the liver through the side port of the endovascular sheath (32); consequently, there is a contraction of remaining liver volume in which the transplanted hepatocytes may engraft limiting the donor cell mass that can be delivered. Volumetric portal embolization (VPE) is a newly described procedure aimed at addressing this barrier to engraftment (71). The procedure, which consists of partial and random distal embolizations of presinusoidal vessels, demonstrated the ability to induce appropriate regenerative signals yet preserved the total liver parenchyma enabling a small long-term increase in the number of transplanted cells and the efficacy of engraftment (71).

To address the problem of cellular engraftment and repopulation of donor hepatocytes after cell transplantation, liver-directed radiation has been suggested as an alternative to facilitate cellular repopulation by inhibiting host hepatocyte proliferation and inducing postmitotic hepatocyte death, allowing donor hepatocytes to preferentially proliferate and repopulate the irradiated host liver (25). The timing of radiation preconditioning of the recipient liver related to HT can also affect engraftment. Delaying HT by 24 hours after preparative hepatic irradiation in mice was shown to enhance engraftment (72) and a shortened interval from irradiation to cell transplant resulted in a lower than anticipated engraftment rate in human studies (25).

Another approach to solve the engraftment and repopulation challenge is by utilizing gene therapy as a tool to select and provide a repopulating advantage (73). Nygaard and colleagues elegantly showed that by introducing a therapeutic transgene (coagulation factor 9) with a selection marker (shRNA, that makes cells resistant to a small-molecule inhibitor of fumarylacetoacetate hydrolase) into transplanted hepatocytes, they could pharmacologically improve the transgene expression in a living animal. These liver preconditioning experiments indicate that it is possible to precondition liver cells using gene-editing approaches to facilitate repopulation of transplanted hepatocytes.

Recent investigations have also interrogated the role of the innate immune system in preventing engraftment. Until hepatocytes traverse the endothelium they remain vulnerable to processes which induce rapid immunological clearance. One such process, the instant blood-mediated inflammatory reaction (IBMIR) occurs in which transplanted hepatocytes are recognized by the innate immune system, leading to activation of both complement and coagulation pathways resulting in cell destruction and low engraftment (74, 75). Importantly, traditional immunosuppression has not been shown to ameliorate the IBMIR (76). Strategies aimed at manipulating the coagulation component (74), the inflammatory component (77), or both in combination (78) of the IBMIR have been shown to improve engraftment.

An alternative approach to prevent activation of the IBMIR following HT is to implant the donor cell mass outside of the portal circulation. One strategy involves the encapsulation of the donor cells in microbeads which provide an immune-protective coating while enabling the maintenance of necessary metabolic functions (75, 79–82). This approach is more amenable to the management of acute liver failure and recent work establishing animal models and developing mechanisms to create banked, cryopreserved microbeads for emergency use are potential advancements (83, 84). Additional techniques used to evade the host immune system include tissue-engineering strategies whereby liver mass constructs are assembled ex vivo and then inserted (usually into the peritoneal cavity) to provide a functional support system (85–87). Unfortunately, these approaches have been limited by overall functional mass efficiency; however, recent advancements have demonstrated higher efficiency as well as a reduction in initial cell function loss (88). Collectively, these advancements make the goal of bioengineered liver units for transplantation potentially more attainable.

Improving Transplanted Hepatocyte Survival and Monitoring

Historically, HT has only partially corrected genetic disorders with the longest registered/published clinical improvement of about 2 years after transplantation (89). A recent report by our group indicates that a combination of radiation preconditioning and segmental portal embolization is effective to improve engraftment and repopulation of transplanted hepatocytes; however, the long-term survival of the graft was hampered by rejection (25). With HT, it is difficult to identify transplanted cells by biopsy, which is routinely performed in solid organ transplant recipients. Thus, it is difficult to know definitively whether the donor cells are being rejected until it is too late to intervene.

Traditional organ transplantation requires regular allograft monitoring for the development of complications such as cell-mediated and antibody-mediated rejection in addition to operational tolerance, all of which assist in the immunosuppressive management of the transplant recipient. Currently, no consensus exists regarding optimal immunosuppressive regimens in HT, with most centers adopting or slightly modifying their institutional protocols for solid organ transplant (1, 2, 5, 6, 12, 15, 26, 29, 90). However, it is becoming clear that the immune responses to HT differ considerably from solid organ transplant and refined immunosuppression strategies are needed to improve the clinical outcome (91).

Cell mediated rejection has been shown to result in allograft loss after liver cell transplantation (2). Tools used in the monitoring of whole or partial liver allografts, such as circulating liver enzymes, are impractical in the setting of HT where only a minority of liver cells constitute the graft (26, 92). Additional serum biomarkers that may be more disease specific, such as bilirubin in Crigler-Najjar or amino acids and ammonia in metabolic diseases, have not been sensitive enough to detect rejection before damage to the allograft is irreversible (25). Routine liver biopsy may be of little use given the random nature of engraftment and the resulting variability in the distribution of donor cells (25, 92). Recent work has proposed that anti-donor activity, as measured by an allospecific CD154+ assay to detect recipient cytotoxic memory T cells, may be a possible strategy to monitor for early evidence of rejection in the setting of HT (25, 93–95). Furthermore, the assay correlated well with a response to a change in therapy, thus potentially identifying a solution to one of the major obstacles in the field of HT (25). Additionally, donor-specific antibody (DSA) driven rejection has recently evolved into an established pathology in solid organ liver transplant (96). The development and contribution of donor-specific antibodies (DSA) in HT is still very much unknown. The presence of de novo DSA following HT has been temporarily associated with graft loss (9, 25) and in one reported case was associated with the peak measurement of the immune reactivity index score that has been shown to enable the prediction rejection (25). Ultimately, a more robust understanding of the immunological responses induced by HT is needed to help guide therapeutic regimens that will enable extended cell graft survival and broader application of HT to patients who will benefit from this promising therapy. Investigators have looked at specific immune stimulatory and inhibitory signals regulating the innate immune response in HT. Researchers investigating the role of CD47, a member of the Ig superfamily which provides a protective signal against phagocytotic activity of macrophages, recently reported on the contribution of donor CD47 in the regulation of T-cell alloresponses in hepatocyte transplantation. Thru a collection of innovative experiments, they were able to show the important role that CD47 plays in the control of not only T-cell alloresponses but also tolerance induction following HT (97). This work, combined with other published works, adds to the building evidence that CD47 incompatibility may induce both innate and adaptive immune mediated rejection of transplanted hepatocytes (97–101).

Additional strategies that are being explored to enable post-transplant cell monitoring include the use of non-invasive imaging techniques such as magnetic resonance imaging (MRI). These MRI-based cell tracking methods have demonstrated the ability to locate nano- or micro-particle tagged hepatocytes in vivo. Importantly, newer technologies, including the use of cells labeled with micron-sized iron oxide particles (MPIO), have shown the capacity to detect single cells by MRI (102–105). Recently, investigators were able to show that porcine hepatocytes could successfully be labeled with MPIO and still maintain their multitude of metabolic functions (106). However, a recurrent challenge is the ability to quantify the number of cells being detected and to confirm that the signal detected is actually an engrafted hepatocyte and not uptake by kuppfer cells or macrophages after hepatocytes have been destroyed. Additional imaging modalities including Cherenkov illumination imaging (CLI), photoacoustic imaging (PAI), surface enhanced Raman imaging (SERI), and theranostic imaging represent further strategies for in vivo cell transplantation monitoring (107–109). Very recently, the thyroidal sodium iodide symporter (NIS) gene was used to visualize transplanted hepatocytes. HCs were transduced ex vivo with the Slc5a5 (NIS) gene under the control of the thyroxine-binding globulin promoter. NIS-transduced hepatocytes could robustly concentrate radiolabeled iodine in vitro. NIS-transduced hepatocytes were readily imaged in vivo by single-photon emission computed tomography, and this demonstrated for the first time noninvasive 3-dimensional imaging of regenerating tissue (110). While challenges such as the capability to assess for viability and safety in tagged cells remain, the ability to monitor cell location after transplant would be an important advancement in the development of human regenerative medicine therapies.

So, What’s next for Clinical Hepatocyte Transplantation?

Clinical trials of hepatocytes transplantation have only demonstrated that is a safe procedure. Questions regarding transplantation of adequate cell numbers to produce clinically satisfactory repopulated liver mass for the different monogenic liver diseases remain. Given the short-term graft survival and immunological hurdles that have been identified in the latest hepatocyte allotransplantation trials, autologous-gene-corrected and fully functional hepatocytes would be ideal. Despite progress in advancing the differentiation of human stem cells into hepatocytes in vitro, cells that replicate the ability of human primary adult hepatocytes to proliferate and completely replace livers for clinical applications has not been achieved. Ultimately, clinical transplantation of autologous liver cells will require the generation of high numbers of liver cells with functionality equal to primary human hepatocytes. Based on the observations that in vivo maturation has been confirmed by genome-wide analysis when human hepatocyte-derived cells were transplanted in the livers of FRG (immunocompromised Fah-deficient mouse) mice for 9 months, it is possible that animals could be used as in vivo bioreactors to mature and biofabricate large amounts of functional human hepatocytes for transplantation. One could imagine future scenarios where liver tissue could be collected from patients with monogenic diseases and gene correction could be achieved in vitro using the CRISPR/CAS9 system. Those cells which successfully underwent functional gene correction could then be selected and amplified in vivo using xenograft animal models to grow hepatocytes. A critical mass of modified, mature hepatocytes would be produced which would enable the original patient/donor to receive an autologous hepatocyte transplantation of their own genetically corrected cells. Hurdles to this approach would need to be overcome, including exposing patients to risky surgical resections and quickening the process of gene correction and selection as isolated human hepatocytes are known to undergo rapid dedifferentiation in vitro.

Several, alternative cell sources including induced pluripotent stem cells (iPSC) and immunomodulatory human amnion epithelial cells (hAEC), as well as others have been developed and studied. (Table 3) To be therapeutically effective, these surrogate hepatocytes must retain the ability to perform the complex metabolic functions and the proliferation capacity of primary human hepatocytes. Ideally, they are also autologous so as to eliminate the need for immunosuppression and the potential for rejection (111).

Table 3.

Alternative sources for future cell-based therapies

Cell Type	Potential Indication	Human studies
Mesenchymal Stem Cells	Liver Failure	Yes
	Liver Cirrhosis
Embryonic Stem Cells	Metabolic Liver Disease	No
	Liver Failure
Induced pluripotent stem cells	Metabolic Liver Disease	No
	Liver Failure
Human amnion epithelial cells	Metabolic Liver Disease	Yes
	Liver Cirrhosis
	Lysosomal Storage Disease

Protocols have been developed to differentiate human pluripotent stem cells into a fetal hepatocyte-like cells (HLC) (112) with studies suggesting that in vivo, these cells could go on to develop a more adult-like phenotype (113–115). Moreover, protocols have been developed that have enabled the reprogramming of somatic cells into inducible pluripotent stem cells (116, 117). The ability to reprogram individual patient’s somatic cells, such as skin fibroblasts, into iPSCs and then re-direct them to develop into HLC has several advantages over other cell sources. These include the generation of an infinitely expandable population of cells and the ability to circumvent immunologic rejection following transplantation. Furthermore, advances in gene editing technologies have facilitated the possibility of correcting the specific genetic anomaly that induce disease and subsequently engineer disease-free autologous cells for re-introduction via HT (118). These technologies are already being tested in murine xenograft models whereby human HLC are being differentiated from iPSCs, engrafted into livers of immunosuppressed mice, and then used to study physiology as well as develop new pharmacologic therapies (119, 120).

hAECs are fetal-derived cells isolated from the amnion membrane in full-term human placentas. Several recent developments have renewed interest in these cells, particularly as it relates to their use in managing liver disease. hAECs are available without the accompanied ethical or religious concerns. Furthermore, in contrast to other sources of pluripotent cells, they do not express telomerase and have not been found to be immortal or tumorigenic when transplanted. They have been reported to express genes normally found in mature liver and, because of an immune-privileged status, might enable the avoidance of immunosuppression in the recipient (121). Prior studies have demonstrated the feasibility of hAEC transplant in patients with lysosomal storage disorders (122–124) and recent work in animal models suggest additional benefit in managing both congenital metabolic disease and liver fibrosis (125).

In summary, the cumulative experience in HT has demonstrated that while partial corrections of metabolic liver disease can be achieved, long-term cures have been elusive. Acute liver failure has been successfully managed with HT and remains an attractive area for therapeutic expansion. Many barriers to HT have been identified and continued scientific and patient directed efforts will be required to identify new cell sources for transplant, enhance engraftment, optimize safety, and improve the outcome thus enabling a broader implementation of this therapy in the treatment of patients with liver disorders.

Abbreviations

OLT

orthotopic liver transplantation

HT

Hepatocyte transplantation

ALF

Acute liver failure

MPIO

micron-sized iron oxide particles

CLI

Cherenkov illumination imaging

PAI

photoacoustic imaging

SERI

surface enhanced Raman imaging

iPSC

inducible pluripotent stem cells

ESC

embryonic stem cells

hAEC

human amnion epithelial cells

MSC

mesenchymal stem cells