Toward Next-Gen Cell Therapy for Pediatric Patients: Neonatal Hepatocytes Tolerate Electroporation-Mediated Gene Editing and Engraft in the Liver.

Neonatal livers are a potential source of good-quality hepatocytes for clinical transplantation. We compared viability and function of neonatal hepatocytes (NHs) and adult hepatocytes (AHs) and report their clinical use both intraportally and in alginate microbeads. Following isolation from donor livers, hepatocyte function was assessed using albumin, alpha-1-antitrypsin, and factor VII. Metabolic function was investigated by measuring resorufin conjugation, ammonia metabolism, uridine diphosphate glucuronosyltransferase enzyme activity, and cytochrome P450 (CYP) function following induction. Activation of the instant blood-mediated inflammatory reaction by NHs and AHs was investigated using an in vitro blood perfusion model, and tissue factor expression was analyzed using real-time polymerase chain reaction (RT-PCR). Clinical hepatocyte transplantation (HT) was undertaken using standard protocols. Hepatocytes were isolated from 14 neonatal livers, with an average viability of 89.4% ± 1.8% (mean ± standard error of the mean) and average yield of 9.3 × 106 ± 2.0 × 106 cells/g. Hepatocytes were isolated from 14 adult livers with an average viability of 78.6% ± 2.4% and yield 2.2 × 106 ± 0.5 × 105 cells/g. NHs had significantly higher viability after cryopreservation than AHs, with better attachment efficiency and less plasma membrane leakage. There were no differences in albumin, alpha-1-antitrypsin, and factor VII synthesis between NHs and AHs (P > 0.05). Neonatal cells had inducible phase 1 enzymes as assessed by CYP function and functional phase 2 enzymes, in which activity was comparable to AHs. In an in vitro blood perfusion model, AHs elicited increased thrombus formation with a greater consumption of platelets and white cells compared with NHs (28.3 × 109 versus 118.7 × 109 and 3.3 × 109 versus 6.6 × 109 ; P < 0.01). Intraportal transplantation and intraperitoneal transplantation of alginate encapsulated hepatocytes was safe, and preliminary data suggest the cells may activate the immune response to a lesser degree than adult cells. In conclusion, we have shown NHs have excellent cell viability, function, and drug metabolism making them a suitable alternative source for clinical HT. Liver Transplantation 24 394-406 2018 AASLD.

Crigler-Najjar type 1 disease is a rare inherited metabolic disease characterized by high levels of unconjugated bilirubin due to the complete absence of hepatic uridine diphosphoglucuronate-glucuronosyltransferase activity. Hepatocyte transplantation (HT) has been proposed as an alternative treatment for Crigler-Najjar syndrome, but it is still limited by the quality and the low engraftment and repopulation ability of the cells used. Because of their attachment capability and expression of adhesion molecules as well as the higher proportion of hepatic progenitor cells, neonatal hepatocytes may have an advantage over adult cells. Adult or neonatal hepatocytes were transplanted into Gunn rats, a model for Crigler-Najjar disease. Engraftment and repopulation were studied and compared by immunofluorescence (IF). Additionally, the serum bilirubin levels, the presence of bilirubin conjugates in rat serum, and the expression of uridine diphosphate glucuronosyltransferase 1 family polypeptide A1 (UGT1A1) in rat liver samples were also analyzed. Here we show that neonatal HT results in long-term correction in Gunn rats. In comparison with adult cells, neonatal cells showed better engraftment and repopulation capability 3 days and 6 months after transplantation, respectively. Bilirubinemia decreased in the transplanted animals during the whole experimental follow-up (6 months). Bilirubin conjugates were also present in the serum of the transplanted animals. Western blots and IF confirmed the presence and expression of UGT1A1 in the liver. This work is the first to demonstrate the advantage of using neonatal hepatocytes for the treatment of Crigler-Najjar in vivo.

Bile acid binding proteins in hepatocellular membranes of newborn and adult rats. Identification of transport proteins with azidobenzamidotauro[ 14C]cholate ([ 14C]ABATC)

Biochemical studies on the age-related toxicity of galactosamine in primary rat hepatocyte cultures

Hepatocyte transplantation is an alternative therapy to orthotopic liver transplantation for the treatment of liver diseases. However, the supply of hepatocytes is limited given the shortage of organs available to isolate good-functioning quality cells. Neonatal livers may be a potential source alternative to adult livers to obtain good-performing hepatic cells for hepatocyte transplantation, which has not yet been explored profoundly. High-yield preparations of viable hepatocytes were isolated from 1- to 23-day-old liver donors, cryopreserved, and banked. Cell integrity and functional quality assessment were performed after thawing. Neonatal hepatocytes showed better postthawing recovery compared with adult hepatocytes, as shown by the viability values that did not differ significantly from freshly isolated cells, a higher expression of adhesion molecules (β1-integrin, β-catenin, and E-cadherin), better attachment efficiency, cell survival, and a lower number of apoptotic cells. The metabolic performance of thawed hepatocytes has been assessed by ureogenesis and drug-metabolizing capability (cytochrome P450 and UDP-glucuronosyltransferase enzymes). CYP2A6, CYP2C9, CYP2E1, and CYP3A4 activities were found in all cell preparations, while CYP1A2, CYP2B6, CYP2C19, and CYP2D6 activities were detected only in hepatocytes from a few neonatal donors. The expression of UGT1A1 and UGT1A9 (transcripts and protein) was detected in all hepatocyte preparations, while activity was measured only in some preparations, probably due to lack of maturity of the enzymes. However, isoforms UGT1A6 and UGT2B7 showed considerable activity in all preparations. Compared to adult liver, the hepatocyte isolation procedure in neonatal livers also provides thawed cell suspensions with a higher proportion of hepatic progenitor cells (EpCAM(+) staining), which could also participate in regeneration of liver parenchyma after transplantation. These results could imply important advantages of neonatal hepatocytes as a source of high-quality cells to improve human hepatocyte transplantation applicability.

Hepatocyte transplantation is a promising treatment for liver failure and inborn metabolic liver diseases, but progress has been hampered by a scarcity of available organs. Here, hepatocytes isolated from livers procured for a neonatal hepatocyte donation program within a research setting were assessed for metabolic function and suitability for transplantation. Organ donation was considered for infants who died in neonatal intensive care in the Stockholm region during 2015–2021. Inclusion was assessed when a decision to discontinue life-sustaining treatment had been made and hepatectomy performed after declaration of death. Hepatocyte isolation was performed by three-step collagenase perfusion. Hepatocyte viability, yield, and function were assessed using fresh and cryopreserved cells. Engraftment and maturation of cryopreserved neonatal hepatocytes were assessed by transplantation into an immunodeficient mouse model and analysis of the gene expression of phase I, phase II, and liver-specific enzymes and proteins. Twelve livers were procured. Median warm ischemia time (WIT) was 190 [interquartile range (IQR): 80–210] minutes. Median viability was 86% (IQR: 71%–91%). Median yield was 6.9 (IQR: 3.4–12.8) x106 viable hepatocytes/g. Transplantation into immunodeficient mice resulted in good engraftment and maturation of hepatocyte-specific proteins and enzymes. A neonatal organ donation program including preterm born infants was found to be feasible. Hepatocytes isolated from neonatal donors had good viability, function, and engraftment despite prolonged WIT. Therefore, neonatal livers should be considered as a donor source for clinical hepatocyte transplantation, even in cases with extended WIT.

Opening a Crack in the Door: An Interview with Haydar Frangoul

Efficient receptor-mediated endocytosis of asialoglycoprotein by mature liver requires participation of microtubules that convey newly internalized ligand to lysosomes for degradation and receptor back to plasma membrane to continue endocytosis. To ascertain whether microtubular participation in asialoglycoprotein endocytosis is altered during development, we compared endocytosis of 125I-labeled asialoorosomucoid (ASOR) in neonatal rat hepatocytes to that in adult cells, with and without microtubular disruption by colchicine. Control experiments demonstrated that 125I-ASOR degradation in neonatal hepatocytes occurred at 70% of the adult rate during continuous endocytosis, although neonatal surface receptors were only approximately 40% as numerous. Colchicine disruption of microtubules reduced 125I-ASOR degradation and steady-state intracellular ASOR more in adults during continuous endocytosis. Degradation of 125I-ASOR prebound to surface receptors was equally impaired by colchicine in the two groups. Continuous ASOR endocytosis by colchicine-treated adult hepatocytes progressively depleted their surface receptors but minimally in neonates. Unlike colchicine, the protonophore monensin markedly impaired receptor recycling as well as postinternalization ligand trafficking in both neonates and adults. Thus these experiments demonstrate that asialoglycoprotein processing proceeds as efficiently in neonatal as in adult hepatocytes despite a reduced surface receptor population. Microtubules are required to maintain receptors on cell surface as well as for postinternalization trafficking in adult cells. During development, only the latter process substantially requires microtubules, indicating that microtubular participation in protein trafficking is selectively, not uniformly, diminished at this time in life.

BackgroundMonoclonal antibody (MCA) therapies have been utilized under emergency use authorization (EUA) for high-risk pediatric patients with mild to moderate coronavirus disease 2019 (COVID-19) in the outpatient setting since late 2019. The purpose of this study was to describe the use of MCA therapy in pediatric patients in the pediatric emergency department (ED) at a large community hospital.MethodsThis was a retrospective case series of high-risk pediatric patients 12 to 17 years of age who received MCA therapy in the pediatric ED between December 8, 2020 and June 3, 2021. The primary outcome was to describe the patient characteristics, clinical presentation, and safety profile of the pediatric population that received MCA therapy. The secondary outcome was to describe the incidence of hospitalizations or ED visits up to 28 days following therapy.ResultsA total of 44 patients were included in the analysis. The median number of days of symptoms was 4 with 41% of patients having symptoms between 0 and 3 days at time of MCA administration. Only one patient experienced a mild adverse event that did not require epinephrine administration. Two patients returned to the ED for reevaluation during the study follow-up period. No patients required admission within 28 days post-therapy.ConclusionsThe administration of MCA therapy in high-risk pediatric patients in the pediatric ED was well-tolerated with subjective improvement noted in COVID-19 symptoms post-therapy. Further studies are necessary to determine the role MCA therapy may play in reducing morbidity from COVID-19 infection in high-risk pediatric patients.

Hepatocyte transplantation

For more than a decade, Luciano Marraffini has played a major role in the CRISPR revolution. As a postdoc with

Successful Half-Dose Busulfan/Full-Dose Fludarabine Based Reduced Intensity Conditioning In High-Risk Pediatric And Adult Chronic Granulomatous Disease (CGD) Patients

Perioperative Echocardiography During the Coronavirus Crisis: Considerations in Pediatrics and Congenital Heart Disease

Clinical pathways are evidence-based tools that have been integrated into many aspects of pediatric hospital medicine and have proven effective at reducing in-hospital complications from a variety of diseases. Adaptation of similar tools for specific, high-risk patient populations in pediatric oncology has been slower, in part due to patient complexities and variations in management strategies. There are few published studies of clinical pathways for pediatric oncology patients. Pediatric patients with a new diagnosis of leukemia or lymphoma often present with one or more "oncologic emergencies" that require urgent intervention and deliberate multidisciplinary care to prevent significant consequences. Here, we present two clinical pathways that have recently been developed using a multidisciplinary approach at a single institution, intended for the care of patients who present with hyperleukocytosis or an anterior mediastinal mass. These clinical care pathways have provided a critical framework for the immediate care of these patients who are often admitted to the pediatric intensive care unit for initial management. The goal of the pathways is to facilitate multidisciplinary collaborations, expedite diagnosis, and streamline timely treatment initiation. Standardizing the care of high-risk pediatric oncology patients will ultimately decrease morbidity and mortality associated with these diseases to increase the potential for excellent outcomes.

Peri-operative management of high-risk paediatric adenotonsillectomy patients: A survey of 35 UK tertiary referral centres