Cell Transplantation


For numerous liver diseases of childhood, liver transplantation (LT) is a lifesaving procedure. However, it requires scarce organs, a highly experienced team to manage the surgical procedure, complications, and follow-up and lifelong immunosuppression for the recipient. Living donor organs and split livers gave us the proof of concept that a partial organ is sufficient to restore liver metabolic functions. Liver cell therapy (LCT), where cells rather than organs are transplanted in the patient, was first evaluated in acute liver failure to support liver function while awaiting spontaneous recovery or as a bridge to transplantation. Recently, LCT has been proposed as a treatment in itself to overcome LT barriers with an off the shelf, easily injectable, and reversible procedure. In addition, it has the advantage of not inducing a strong immunogenic response. Initially, LCT was performed for liver-based inborn errors of metabolism (IEM) with hepatocytes isolated from livers not suitable for LT, but stem cells are increasingly of interest for acquired liver diseases.

Hepatocyte Transplantation

Clinical Application

Hepatocyte transplantation (HT) was translated to human medicine in the 1990s to overcome the limitations of LT—lack of donors, intensive surgery, cost, immunosuppression. In 1994, Habibullah et al. reported on intraperitoneal fetal hepatocytes administration in seven patients with acute liver failure; one child was included in the study and survived the acute decompensation. The next year, Grossman et al. published the first intraportal injection of autologous hepatocytes transduced with a low-density lipoprotein (LDL) receptor in five patients (three children) with familial hypercholesterolemia; LDL cholesterol decreased in three of them. The autologous procedure was developed to avoid immunosuppression and the allogenic variable. Finally, in 1997, an intraportal heterologous hepatocyte infusion in a 5-year-old boy diagnosed with ornithine transcarbamylase (OTC) deficiency was reported. His ammonia and glutamine levels returned to normal values at discharge. Unfortunately, the child died 43 days post-HT of liver biopsy complications. Since the first report in 1994, HT has been evaluated in 58 children for indications ranging from biliary atresia by acute liver failure ( Table 32.1 ) to liver-based IEM ( Table 32.2 ), with variable clinical success. At least 52% of them (30/58) received LT after HT. Currently, HT may be considered a bridge to transplantation, especially for patients with liver-based IEM who usually face a long waiting time before LT.

Table 32.1

Clinical Hepatocyte Transplantation in Pediatric Acute Liver Failure

(From Gramignoli R, et al. Clinical hepatocyte transplantation: practical limits and possible solutions. Eur Surg Res. 2015;54(3-4):162-177; Puppi J, Dhawan A. Human hepatocyte transplantation overview. Methods Mol Biol. 2009;48:11-16; Hansel MC, et al. The history and use of human hepatocytes for the treatment of liver diseases: the first 100 patients. Curr Protoc Toxicol. 2014;62:14.12.1-23; Khan Z, Strom SC. Hepatocyte transplantation in special populations: clinical use in children. Methods Mol Biol. 2017;1506:3-16.)

Cause Age Effect, Outcome Reference
Drug-induced 16 years
12 years
10 years
Ammonia reduction, death, 2 days post-HT
Ammonia reduction, death, 7 days post-HT
Ammonia reduction, death, 7 days post-HT
6 months Ammonia reduction, life support withdrawal and death, 7 days post-HT
13 years Death, 4 days post-HT
14 years Ammonia reduction and improved encephalopathy, LT 1 day post-HT
Idiopathic 8 years Intraperitoneal injection of fetal hepatocytes, full recovery
3 years
5 years
Ammonia reduction and improved encephalopathy in both
Full recovery and immunosuppression weaned; successful bridge to LT 4 days post-HT
3.5 months No clear benefit, LT 1 day post-HT
Virus-induced 4 years Ammonia reduction and improved encephalopathy, intracranial hypertension on day 2
3 weeks Ammonia reduction, death, 11 days post-HT

HT, Hepatocyte transplantation; LT, liver transplantation.

Table 32.2

Clinical Hepatocyte Transplantation in Pediatric Liver-Based Inborn Errors of Metabolism

(From Gramignoli R, et al. Clinical hepatocyte transplantation: practical limits and possible solutions. Eur Surg Res. 2015;54(3-4):162-177; Pareja E, Gomez-Lechon ML, Tolosa L. Alternative cell sources to adult hepatocytes for hepatic cell therapy. Methods Mol Biol. 2017;1506:17-42; Puppi J, Dhawan A. Human hepatocyte transplantation overview. Methods Mol Biol. 2009;48:11-16; Hansel MC, et al. The history and use of human hepatocytes for the treatment of liver diseases: the first 100 patients. Curr Protoc Toxicol. 2014;62:14.12.1-23; Khan Z, Strom SC. Hepatocyte transplantation in special populations: clinical use in children. Methods Mol Biol. 2017;1506:3-16.)

Cause Age Effect, Outcome Reference
Crigler-Najjar syndrome type 1 10 years 50% reduction in bilirubin, reduction in phototherapy, LT 4 years post-HT
8 years 40% reduction in bilirubin, LT 20 months post-HT
9 years 30% reduction in bilirubin, 35% reduction in phototherapy, LT 5 months post-HT
1.5 years

3 years
> 50% reduction in bilirubin, reduction in phototherapy, LT 8 months post-HT
30% reduction in bilirubin, LT 18 months post-HT
3.5 years Lowered serum bilirubin, outcome unknown
8 years 35% reduction in bilirubin, 50% reduction in phototherapy, LT 11 months post-HT
9 years
1 year
20% reduction in bilirubin, LT 6 months post-HT
25% reduction in bilirubin, LT 4 months post-HT
2 years 50% reduction in bilirubin, outcome unknown
11 years 20% reduction in bilirubin, LT waiting list
7 months 50% reduction in bilirubin and in phototherapy, psychomotor improvement, bilirubin stable at 1-year follow-up
13 years

11 years
50% reduction in bilirubin, presence of bile glucuronides in bile, LT 19 months post-HT
50% reduction in bilirubin, presence of bile glucuronides in bile, LT 31 months post-HT
Alpha-1 antitrypsin deficiency 18 weeks LT 2 days post-HT, cirrhosis on explant
Familial hypercholesterolemia 12 years
7 years
11 years
Ex vivo gene therapy with autologous cells.
No benefit; 6% reduction in total cholesterol and LDL cholesterol
19% reduction in total cholesterol and LDL cholesterol
12 years 13% reduction in total cholesterol and LDL cholesterol
Factor VII deficiency 3 months
35 months
70% reduction in rFVII requirement, LT 7 months post-HT
70% reduction in rFVII requirement, LT 8 months post-HT
4 months Reduction in rFVII requirement, outcome unknown
Progressive familial intrahepatic cholestasis type 2 32 months
16 months
No benefit (cirrhosis established):, LT 5 months post-HT
No benefit (cirrhosis established), LT 14 months post-HT
Phenylketonuria 6 years Reduction in phenylalanine levels and improved dietary tolerance up to 3 months post-HT (cells from “domino” GSD1b liver), PAH activity on liver biopsy at 11 months post-HT
Tyrosinemia type 1 59 days Improved coagulopathy and bilirubin, LT 45 days post-HT (cirrhosis on explant)
Glycogen storage disease type 1a 6 years Reduction in hypoglycemic episodes and cholesterol and triglycerides levels, no hypoglycemic admission at 1-year follow-up
Glycogen storage disease type 1b 18 years Improved blood glucose, decreased epistaxis, normal G6Pase activity on liver biopsy at 8 months post-HT
Mild Zellweger spectrum disorder 4 years 40% reduction in pipecolic acid for 18 months, decreased cholestasis and abnormal bile acid, psychomotor improvement, outcome unknown
Primary hyperoxaluria type 1 15 months Reduction un plasma oxalate, liver-kidney transplant 13 months post-HT
Urea cycle defects
Ornithine transcarbamylase deficiency 5 years Ammonia reduction and protein tolerance, death by sepsis 43 days post-HT
5 years Ammonia reduction, normal glutamine, death 45 days post-HT
10 hours Ammonia reduction and protein tolerance, LT 6 months post-HT
1 day Ammonia reduction, increased urea, protein tolerance, auxiliary partial LT 7 months post-HT and neurologically normal
14 months Ammonia reduction, increased urea, psychomotor improvement, LT 6 months post-HT ,
1 day Ammonia reduction, increased urea, protein tolerance, auxiliary partial LT 7 months post-HT
6 hours

9 days
Ammonia reduction, increased urea, normal urine orotic acid, death 4 months post-HT
Ammonia reduction, protein tolerance, normal urine orotic acid, LT waitlist 6 months post-HT
12 years Ammonia reduction, increased urea, normal glutamine, septic death 30 days post-HT
11 days Ammonia reduction, neurologically normal 3 months post-HT
7 months No effect, LT 4 months post-HT
Argininosuccinate lyase deficiency 3.5 years Ammonia reduction, psychomotor improvement, LT 18 months post-HT ,
Carbamoyl phosphate synthase I deficiency 2.5 months Ammonia reduction and increased urea, LT 15 months post-HT ,
4 months No effect, LT 3.5 months post-HT
Citrullinemia 25 months Ammonia reduced and increased urea, outcome unknown (Lee et al., unpublished)
3 years Ammonia reduction, increased urea, protein tolerance, outcome unknown

G6Pase, Glucose-6-phosphatase; GSD1b, glycogen storage disease type 1b; HT, hepatocyte transplantation; LDL, low-density lipoprotein; LT, liver transplantation; rFVII, recombinant factor VII; PAH, phenylalanine hydroxylase.

Material Source

The first HT procedure was performed with using fetal hepatocytes, generating some ethical considerations. One team used magnetic activated cell sorting to purify hepatic progenitor cells from fetal hepatocyte based on the CD326 expression.

Today, the main source of cells for HT are the livers unsuitable for LT, such as reduction remnants or unused split livers, damaged livers, or livers from young donors. Steatotic livers are of lesser quality for hepatocytes. Hepatocytes seem to tolerate ischemia well in comparison with cholangiocytes. Yet, their viability has been shown to be inversely correlated to ischemia time. Hepatocytes can also be isolated from segment IV, with or without caudate lobe during a split-liver procedure or from non-heart-beating donors. As for domino liver transplantation, hepatocytes collected from an explanted liver affected by a specific IEM are suitable for HT in patients with another IEM. A 6-year-old child with tetrahydrobiopterin unresponsive phenylketonuria received hepatocytes isolated from the native liver of a patient transplanted for glycogen storage disease type 1b. Phenylalanine levels returned to normal, and their half-life decreased significantly after the procedure. To date, no difference in clinical outcome has been reported based on the hepatocyte origin.

Hepatocyte Isolation

A two-step collagenase digestion procedure was developed by Seglen to obtain hepatocytes from rat liver. The procedure was adapted to the human liver by Strom et al. and since has remained the standard protocol to isolate human hepatocytes. Briefly, the liver is perfused with a buffered solution containing a calcium-chelating agent to loosen the desmosomal junctions. Then, collagenase is infused into the liver via the cannulated hepatic veins. After isolation, hepatocytes are usually cryopreserved to be available off the shelf. Unfortunately, this affects their viability after thawing. The whole hepatocyte isolation process has to be done under a laminar flow in a clean room, with regular bacterial and fungus checking. Moreover, because HT falls under advanced therapy medicinal product laws, the US Food and Drug Administration and European Medicines Agency require compliance with the guidelines for Good Manufacturing Practice.

Quality Control

Hepatocytes must be tested for bacterial contamination and Mycoplasma . A Trypan blue exclusion test is usually performed to quantify cell viability before and after infusion and after thawing in case of cryopreservation Viability in excess of 60% is required to use for cells for clinical application. To evaluate cell engraftment potential, hepatocytes are plated on collagen-coated dishes for 24 hours, and then adherent cells are counted and the ratio to seeded cells number calculated. Recently, an assay comprised of 11 end points was developed to quantify the metabolic capacity of isolated hepatocytes, but its clinical relevance has yet to be demonstrated.

Dose and Route of Administration

A 70-kg adult liver is estimated to contain 2.8 × 10 11 hepatocytes, or 4 × 10 9 cells/kg of body weight. HT aims to replace 2% to 5% of the patient’s liver mass to restore substantial metabolic liver function, which is equivalent to 8 to 20 × 10 7 cells/kg. This number of cells is usually administered in multiple infusions to avoid the risk of portal thrombosis, which may be linked to the procoagulant activity of hepatocytes. In patients with liver-based IEM, HT can be repeated in case of loss of effect over time.

The first and only report of HT delivered through intraperitoneal infusion was for acute liver failure, because this route allows severely ill patients to benefit from HT. Intrasplenic arterial infusions have also been evaluated in patients with altered liver architecture and coagulation problems. Intraportal infusions are the most common way used to deliver cells to the recipient. During the infusion, the portal pressure and vital signs must be monitored regularly. Indium-111-labeled hepatocytes were intraportally infused in a 5-year-old child with OTC deficiency; it was shown the cells are preferentially retained in the recipient liver.

Engraftment Evaluation and Enhancement

Hepatocyte engraftment quantification has been performed on liver biopsies with the limitations of sampling, meaning that the quantification is based on a small fraction of the whole organ, limiting background extrapolation and, more accurately, on explanted livers from patients undergoing LT after HT. In donor-recipient sex mismatch, engraftment can be extrapolated from sex-determining region Y gene quantification by quantitative polymerase chain reaction. However, this technique is limited by the background signal coming from apoptotic cells and cell debris. The enzyme activity quantification on a liver sample is clinically more relevant marker of engraftment in liver-based IEM. In these diseases, the effect of HT is assessed by observing the decrease of an accumulating compound (e.g., ammonia in urea cycle disorders, UCDs) or the appearance of a downstream product (e.g., urea in UCD or conjugated bilirubin in Crigler-Najjar syndrome type 1 [CN1] ).

Partial hepatectomy (PH) is regularly performed for living donor liver harvesting or liver tumor resection. This induces a strong stimulus for hepatocyte replication. PH has been used in HT to stimulate proliferation of infused hepatocytes given that, in the absence of a selective advantage, donor and recipient cells proliferate at the same rate. For example, preoperative portal vein occlusion (PVO) is a common surgical procedure to induce hepatic regeneration before PH for tumor resection. To our knowledge, this technique has never been applied in HT. Taken together, PH and PVO could make HT a more complex procedure, and the risk-benefit ratio would need to be carefully evaluated.

Irradiation of the native liver has been developed to induce a strong mitogenic signal in the liver parenchyma with a proliferative advantage of the (nonirradiated) infused hepatocytes without the potential risks of PH. Recently, Soltys et al. reported HT in two pediatric patients (aged 4 and 7 months, respectively) with UCD preconditioned with radiation therapy to the right lobe of the liver (5 and 7.5 Gy, respectively). HT had no effect on metabolic control, and both patients underwent LT a few months after HT. The explanted livers were not screened for infused cells and showed no sign of radiation-induced damage. Yet, liver irradiation is not without side effects; it was shown in six adult patients treated with high-dose radiation (12-54 Gy) for biliopancreatic carcinoma, to activate stellate cells with the risk of liver fibrosis in the long term,.


Immunosuppression regimens following HT have been largely inspired by LT protocols, including induction based on basiliximab at days 0 and 4 and maintenance therapy with tacrolimus to keep the serum levels at 6 to 8 ng/mL. Some have added, as induction regimen, intravenous methylprednisolone followed by prednisolone daily tapered over the first 6 months post-HT. Recently, a patient received antilymphocyte globulin in attempt to control. To target rejection of the infused hepatocytes and functional loss. Avoid rejection of the infused hepatocytes, Grossman et al. used autologous transduced hepatocytes to treat patients with familial hypercholesterolemia, but without much clinical success.

How to Overcome Hepatocyte Transplantation Barriers

Since the first report of HT nearly 25 years ago, much effort has been focused on how to translate HT into a validated clinical option. Hepatocytes are fragile cells with low proliferative capacities in vitro , and their engraftment is low, even with intense preconditioning methods, such as radiation therapy associated with strong immunosuppression (antilymphocyte antibody). The field of liver cell therapy is now looking for a new type of cell that can overcome the disadvantages of a fully differentiated primary cell. Mesenchymal stromal cells (MSCs) or, as recently renamed, medicinal signaling cells, seem to hold promise for clinical application in a near future.

Stem and Progenitor Cell-Based Cell Transplantation

Stem and Progenitor Cells and Their Regenerative Potential

Stem cells are defined as unspecialized cells demonstrating self-renewal capacity, a high proliferative potential, and the ability to differentiate into multiple specialized cell types. This proliferative potential is conserved during in vitro cultures and, along with their fairly robust resistance to cryopreservation, offers a major advantage over hepatocytes ( Table 32.3 ).

Feb 23, 2021 | Posted by in HEPATOPANCREATOBILIARY | Comments Off on Cell Transplantation

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