Liver cell transplantation for Crigler-Najjar syndrome type Ⅰ： Update and perspectives

Liver cell transplantation is an attractive technique to treat liver-based inborn errors of metabolism. The feasibility and efficacy of the procedure has been demonstrated, leading to medium term partial metabolic control of various diseases. Crigler-Najjar is the paradigm of such diseases in that the host liver is lacking one function with an otherwise normal parenchyma. The patient is at permanent risk for irreversible brain damage. The goal of liver cell transplantation is to reduce serum bilirubin levels within safe limits and to alleviate phototherapy requirements to improve quality of life. Preliminary data on Gunn rats, the rodent model of the disease, were encouraging and have led to successful clinical trials. Herein we report on two additional patients and describe the current limits of the technique in terms of durability of the response as compared to alternative therapeutic procedures. We discuss the future developments of the technique and new emerging perspectives.     

Hepatocyte transplantation: State of the art

Advances in biotechnology have allowed hepatocyte transplantation as a relevant proposition to treat liver disease. This procedure may change the crescent mortality in liver transplantation waiting lists due global organ shortage. Recent clinical trials have described promising results of hepatocyte transplantation for acute, acute-on-chronic and metabolic liver disease. In this report, we discuss progresses regarding hepatocyte culture, cryopreservation systems, hepatocyte immortalization, suitable recipient site for hepatocyte engraftment, cell differentiation and fusion into hepatocytes, current clinical trials, and summarize the bioartificial liver systems. These progressions motivate expectation concerning hepatocyte transplantation as a consistent therapy for liver disease.     

成人原代肝细胞的分离、培养及冻存

目的:建立一种稳定的成人原代肝细胞分离、培养、冻存方法,为肝细胞移植、生物人工肝支持系统治疗急慢性肝病以及肝细胞体外应用模型提供潜在的肝细胞资源.方法:20例供肝采用离体两步胶原酶灌注技术分离成人原代肝细胞.选择7个不同的预培养时间点(2、6、12、24、36、48和72h),分离所获肝细胞按上述不同预培养时间在4℃人无血清培养基(HepatoZYME-SFM)中培养,然后收集预培养肝细胞转移到含100mL/L胎牛血清和DMSO的HepatoZYME-SFM中,再立即放入-80℃异丙醇冷冻盒过夜,次日投入液氮.分析比较各肝细胞在解冻后细胞活力率、贴壁率、白蛋白分泌及尿素合成.结果:在部分肝叶切除后使用离体两步胶原酶灌流技术分离所得肝细胞活率和贴壁率分别是75.0%±4.6%和72.0%±6.0%.4℃预培养12或24h被证明是最适预培养时间,这两个时间点的白蛋白分泌高于其他时间点(P<0.05).与立即冷冻组相比较,预培养12或24h肝细胞解冻后活力(61.4%±4.8%,62.0%±5.6%vs53.4%±4.2%)、贴壁率(63.2%±5.8%,62.6%±3.6%vs55.2%±4.6%)、白蛋白分泌及...     

Hepatocytes isolated from neoplastic liver-immunomagnetic purging as a new source for transplantation

AIM： To investigate whether hepatocytes isolated from macroscopically normal liver during hepatic resection for neoplasia could provide a novel source of healthy hepatocytes, including the development of reliable protocols for malignant cells removal from the hepatocyte preparation. METHODS： Hepatocytes were procured from resected liver of 18 patients with liver tumors using optimised digestion and cell-enrichment protocols. Suspensions of various known quantities of the HT-29 tumor cell line and patient hepatocytes were treated or not with Ep-CAM-antibody-coated immunomagnetic beads in order to investigate the efficacy of tumor-purging by immunomagnetic depletion, using a semi-quantitative RT-PCR method developed to detect tumor cells. Immunomagnetic bead-treated or bead-untreated tumor cell-hepatocyte suspensions were transplanted intra-peritoneally in Balb/C nude mice to assess the rates of tumor development. RESULTS： Mean viable hepatocyte yield was 9.3×10^6 cells per gram of digested liver with mean viability of 70.5%. Immunomagnetic depletion removed tumor cells to below the RT-PCR detection-threshold of 1 tumor cell in 10^6 hepatocytes, representing a maximum tumor purging efficacy of greater than 400000-fold. Transplanted, immunomagnetic bead-purged tumor cell-hepatocyte suspensions did not form peritoneal tumors in Balb/C nude mice. Co-transplantation of hepatocytes with tumor cells did not increase tumorigenesis of the tumor cells. CONCLUSION： Immunomagnetic depletion appears to be an effective method of purging contaminating tumor cells to below threshold for likely tumorigenesis. Along with improved techniques for isolation of large numbers of viable hepatocytes, normal liver resected for neoplasia has potential as another clinically useful source of hepatocytes for transplantation.     

Transplantation in inborn errors of metabolism: current considerations and future perspectives

Inborn errors of metabolism (IEM) comprise an assorted group of inherited diseases, some of which are due to disordered lysosomal or peroxisomal function and some of which might be improved following haemopoietic cell transplantation (HCT). In these disorders, the onset in infancy or early childhood is typically accompanied by rapid deterioration, resulting in early death in the more severe phenotypes. Timely diagnosis and immediate referral to an IEM specialist are essential steps in optimal management. Treatment recommendations are based on the diagnosis, its phenotype, rate of progression, prior extent of disease, family values and expectations and the risks and benefits associated with available therapies, including HCT. International collaborative efforts are of utmost importance in determining outcomes of therapy for these rare diseases, and have improved those outcomes significantly over recent decades. This discussion focusses on HCT in IEM, providing an international perspective on progress, limitations, and future directions.     

Hepatocyte isolation from resected benign tissues: Results of a 5-year experience

AIM To analyze retrospectively a 5-year experience of human hepatocyte isolation from resected liver tissues with benign disease.METHODS We established a method of modified four-step retrograde perfusion to isolate primary human hepatocytes. Samples were collected from the resected livers of patients with intrahepatic duct calculi(n = 7) and liver hemangioma(n = 17). Only the samples weighing ≥ 15 g were considered suitable for hepatocyte isolation. By using the standard trypan blue exclusion technique, hepatocyte viability and yield were immediately determined after isolation.RESULTS Twenty-four liver specimens, weighing 15-42 g, were immediately taken from the margin of the removed samples and transferred to the laboratory for hepatocyte isolation. Warm ischemia time was 5-35 min and cold ischemia time was 15-45 min. For the 7 samples of intrahepatic duct calculi, the method resulted in a hepatocyte yield of 3.49 ± 2.31 × 10~6 hepatocytes/g liver, with 76.4% ± 10.7% viability. The 17 samples of liver hemangioma had significantly higher yield of cells(5.4 ± 1.71 × 10~6 cells/g vs 3.49 ± 2.31 × 10~6 cells/g, P 0.05) than the samples of intrahepatic duct calculi. However, there seems to be no clear difference in cell viability(80.3% ± 9.67% vs 76.4% ± 10.7%, P 0.05). We obtained a cell yield of 5.31 ± 1.87 × 10~6 hepatocytes/g liver when the samples weighed 20 g. However, for the tissues weighing ≤ 20 g, a reduction in yield was found(3.08 ± 1.86 × 10~6 cells/g vs 5.31 ± 1.87 × 10~6 cells/g, P 0.05).CONCLUSION Benign diseased livers are valuable sources for largenumber hepatocyte isolation. Our study represents the largest number of primary human hepatocytes isolated from resected specimens from patients with benign liver disease. We evaluated the effect of donor liver characteristics on cell isolation, and we found that samples of liver hemangioma can provide better results than intrahepatic duct calculi, in terms of cell yield. Furthermore, the size of the tissues can affect the outcome of hepatocyte isolation.     

Alginate microencapsulated human hepatocytes for the treatment of acute liver failure in children

1. Download : Download high-res image (179KB)
2. Download : Download full-size image

     

Hepatocyte cell therapy in liver disease

Liver disease is a leading cause of morbidity and mortality. Liver transplantation remains the only proven treatment for end-stage liver failure but is limited by the availability of donor organs. Hepatocyte cell therapy, either with bioartificial liver devices or hepatocyte transplantation, may help address this by delaying or preventing liver transplantation. Early clinical studies have shown promising results, however in most cases, the benefit has been short lived and so further research into these therapies is required. Alternative sources of hepatocytes, including stem cell-derived hepatocytes, are being investigated as the isolation of primary human hepatocytes is limited by the same shortage of donor organs. This review summarises the current clinical experience of hepatocyte cell therapy together with an overview of possible alternative sources of hepatocytes. Current and future areas for research that might lead towards the realisation of the full potential of hepatocyte cell therapy are discussed.     

Liver transplantation for alcoholic liver disease: Lessons learned and unresolved issues

The use of liver transplantation(LT) as a treatment for alcoholic liver disease(ALD) has been highly controversial since the beginning. The ever increasing shortage of organs has accentuated the low priority given to patients suffering from ALD, which is considered a "self-inflicted" condition. However, by improving the long-term survival rates, making them similar to those from other indications, and recognizing that alcoholism is a primary disease, ALD has become one of the most common indications for LT in Europe and North America, a situation thought unfathomable thirty years ago. Unfortunately, there are still many issues with the use of this procedure for ALD. There are significant relapse rates, and the consequences of excessive drinking after LT range from asymptomatic biochemical and histological abnormalities to graft failure and death. A minimum three-month period of sobriety is required for an improvement in liver function, thus making LT unnecessary, and to demonstrate the patient's commitment to the project, even though a longer abstinence period does not guarantee lower relapse rates after LT. Recent data have shown that LT is also effective for severe alcoholic hepatitis when the patient is unresponsive to corticosteroids therapy, with low relapse rates in highly selected patients, although these results must be confirmed before LT becomes a standard procedure in this setting. Finally, LT for ALD is accompanied by an increased risk of de novo solid organ cancer, skin cancer, and lymphoproliferative disorders, which has a large impact on the survival rates.     

Hepatocyte transplantation: new horizons and challenges

Hepatocyte transplantation represents an alternative strategy for treating liver disease. Liver repopulation following acute liver failure could, potentially, eliminate the requirement for orthotopic liver transplantation. Similarly, the ability to repopulate the liver with disease-resistant hepatocytes offers new opportunities for correcting genetic disorders and treating patients with chronic liver disease. Recent advances concerning the fate of transplanted cells in the recipient liver, the efficacy of cell therapy in outstanding animal models of human disease, and the isolation of progenitor liver cells capable of differentiating into mature hepatocytes have renewed optimism in regard to treating people with hepatocyte transplantation. Recruitment of an increasing number of investigators to the field and the success of recent pilot studies indicate that hepatocyte transplantation will become routine clinical practice in the near future.     

Hepatocyte and Kupffer cell function after liver transplantation in the rat –in vivo evaluation with dynamic scintigraphy

Abstract: In vivo physiological measurements of hepatocyte and Kupffer cell function after liver transplantation are desirable. Orthotopic liver transplantation was performed in 54 rats. Hepatocyte and Kupffer cell function were measured with dynamic liver scintigraphy. Hepatic clearance of ^99mTc-Nanocoll (%/min), an albumin colloid phagocytosed by the Kupffer cells, was used to evaluate Kupffer cell function. Hepatic clearance of ^99mTc-IODIDA (%/min), an imino-diacetic-acid taken up and secreted by the hepatocytes, was used to evaluate the hepatocyte function. Hepatic clearance in control rats was 27±2 %/min for Nanocoll and 30±3 %/min for IODIDA. After syngenic liver transplantation, without rejection, there was a rise in Nanocoll clearance (34±2 %/min p<0.01) after 3 weeks, but no change in IODIDA clearance (32±3 %/min N.S.). After syngenic liver transplantation with preservation time prolonged to 16 h, there were no changes in IODIDA or Nanocoll clearance 1 day after transplantation. Both IODIDA (11±2 %/min) and Nanocoll clearance (22±2 %/min) were decreased (p<0.001) during rejection after allogenic transplantation. An in vivo method of measuring the hepatocyte and Kupffer cell function in the transplanted liver is described. Kupffer cell function was increased after syngenic liver transplantation. Kupffer cell and hepatocyte function were decreased during rejection. Dynamic liver scintigraphy seems a suitable procedure for examining liver injury after liver transplantation in the experimental setting.     

Early liver transplantation is essential for familial amyloidotic polyneuropathy patients' quality of life

Nineteen patients, who had undergone liver transplantation for familial amyloidotic polyneuropathy, had answered a quality of life questionnaire including 61 questions on somatic and mental symptoms, social aspects of life, confidence and satisfaction before, one year, and two years after transplantation.

We found that patient satisfaction was generally good two years or more after the transplantation. Most of the patients were very or quite satisfied with the result. All of them had the drive to go on and felt hopeful about the future. However, on the second follow-up, 37% of the patients noted that they felt more insecure in their everyday life and there was a significant difference between the two assessments. The diarrhea score became worse between one and two years after the transplantation and was closely related to the duration of the gastrointestinal symptoms and to the duration of the disease before transplantation. The mental symptoms also increased significantly between the evaluations and this related to the severity of the somatic symptoms.

Our conclusion is that liver transplantation should be performed before advanced somatic symptoms start to develop in order to improve the patients' chances of a good quality of life following liver transplantation.     

儿童肝移植的现状和未来

儿童肝移植已经成为儿童终末期肝病的标准治疗方法。发展儿童肝移植意义重大。本文从适应证、生存率、手术方式、技术性并发症、免疫抑制治疗、远期生存状况和受体危险度分层等方面对目前儿童肝移植的发展水平进行初步概述,并对未来发展作初步展望。     

Hepatocellular carcinoma Liver Imaging Reporting and Data Systems treatment response assessment: Lessons learned and future directions

Hepatocellular carcinoma(HCC) is a leading cause of morbidity and mortality worldwide, with rising clinical and economic burden as incidence increases. Thereare a multitude of evolving treatment options, including locoregional therapies which can be used alone, in combination with each other, or in combination with systemic therapy. These treatment options have shown to be effective in achieving remission, controlling tumor progression, improving disease free and overall survival in patients who cannot undergo resection and providing a bridge to transplant by debulking tumor burden to downstage patients. Following locoregional therapy(LRT), it is crucial to provide treatment response assessment to guide management and liver transplant candidacy. Therefore, Liver Imaging Reporting and Data Systems(LI-RADS) Treatment Response Algorithm(TRA) was created to provide a standardized assessment of HCC following LRT. LIRADS TRA provides a step by step approach to evaluate each lesion independently for accurate tumor assessment. In this review, we provide an overview of different locoregional therapies for HCC, describe the expected post treatment imaging appearance following treatment, and review the LI-RADS TRA with guidance for its application in clinical practice. Unique to other publications, we will also review emerging literature supporting the use of LI-RADS for assessment of HCC treatment response after LRT.     

Impact of gastrointestinal dysfunction on survival after liver transplantation for familial amyloidotic polyneuropathy

Liver transplantation is the only effective treatment of familial amyloidotic polyneuropathy type I (FAP). The aim of the present investigation was to identify factors at the time of submission for transplantation that had impact on survival, with special reference to gastrointestinal disturbances. All 28 liver-transplanted FAP patients evaluated at Umeå University Hospital were included in the study. A modified body mass index was used to assess nutritional status. Intestinal examinations were performed to diagnose bile acid malabsorption, gastric retention, and bacterial contamination of the small bowel. A significantly improved survival rate was found for patients in a good nutritional state (P=0.002). Peripheral neurological symptoms were unrelated to survival, whereas increased mortality was found for patients with bile acid malabsorption (P<0.05). Bacterial contamination and gastric retention were common complications of the disease. In conclusion, malabsorption and malnutrition have a profound impact on the outcome of liver transplantation for familial amyloidotic polyneuropathy.The study was supported by grants from the patients association FAMY, Umeå Health District, the Swedish Medical Research Council, grant 19X-11240, Umeå University and the Joint Committee of the Northern Health Districts.     

Treating diabetes with islet transplantation: Lessons from the past decade in Lille

Type 1 diabetes (T1D) is due to the loss of both beta-cell insulin secretion and glucose sensing, leading to glucose variability and a lack of predictability, a daily issue for patients. Guidelines for the treatment of T1D have become stricter as results from the Diabetes Control and Complications Trial (DCCT) demonstrated the close relationship between microangiopathy and HbA1c levels. In this regard, glucometers, ambulatory continuous glucose monitoring, and subcutaneous and intraperitoneal pumps have been major developments in the management of glucose imbalance. Besides this technological approach, islet transplantation (IT) has emerged as an acceptable safe procedure with results that continue to improve. Research in the last decade of the 20th century focused on the feasibility of islet isolation and transplantation and, since 2000, the success and reproducibility of the Edmonton protocol have been proven, and the mid-term (5-year) benefit–risk ratio evaluated. Currently, a 5-year 50% rate of insulin independence can be expected, with stabilization of microangiopathy and macroangiopathy, but the possible side-effects of immunosuppressants, limited availability of islets and still limited duration of insulin independence restrict the procedure to cases of brittle diabetes in patients who are not overweight or have no associated insulin resistance. However, various prognostic factors have been identified that may extend islet graft survival and reduce the number of islet injections required; these include graft quality, autoimmunity, immunosuppressant regimen and non-specific inflammatory reactions. Finally, alternative injection sites and unlimited sources of islets are likely to make IT a routine procedure in the future.     

Islet-after-failed-pancreas and pancreas-after-failed islet transplantation: Two complementary rescue strategies to control diabetes

For selected patients with type 1 diabetes, β-cell replacement is the treatment of choice, either by islet transplantation (ITX) or whole pancreas transplantation (PTX). When either modality fails, current practice is to consider retransplantation, or return to exogenous insulin. We investigate outcomes with PTX after failed ITX (PAI), and ITX after failed PTX (IAP). All patients receiving PAI or IAP at a single institution were identified. Donor and recipient variables were documented, including transplant outcomes analyzed for insulin requirement and metabolic control. Five subjects were listed for PAI, and 2 received transplants. Of the 4 listed for IAP, 3 have received transplants. The mean waitlist time was 4.5 ± 4.1 y for PAI and 0.35 ±0 .4 y for IAP (p = 0.08). Metabolic control was excellent after PAI, with 2/2 insulin-independent. After IAP, 1/2 achieved insulin independence and good metabolic control after 2 islet infusions. The third could not receive 2^nd infusion and presented c-peptide levels < 0.1 nmol/L. Both strategies are feasible. The outcomes after PAI in our center must be offset by much longer waitlist time due to the sensitization status of these patients. Data from multicentre experience will allow more robust comparative outcomes to be made, the current observations being restricted to a limited patient set.     

Islet-after-failed-pancreas and pancreas-after-failed islet transplantation: Two complementary rescue strategies to control diabetes

For selected patients with type 1 diabetes, β-cell replacement is the treatment of choice, either by islet transplantation (ITX) or whole pancreas transplantation (PTX). When either modality fails, current practice is to consider retransplantation, or return to exogenous insulin. We investigate outcomes with PTX after failed ITX (PAI), and ITX after failed PTX (IAP). All patients receiving PAI or IAP at a single institution were identified. Donor and recipient variables were documented, including transplant outcomes analyzed for insulin requirement and metabolic control. Five subjects were listed for PAI, and 2 received transplants. Of the 4 listed for IAP, 3 have received transplants. The mean waitlist time was 4.5 ± 4.1 y for PAI and 0.35 ±0 .4 y for IAP (p = 0.08). Metabolic control was excellent after PAI, with 2/2 insulin-independent. After IAP, 1/2 achieved insulin independence and good metabolic control after 2 islet infusions. The third could not receive 2^nd infusion and presented c-peptide levels < 0.1 nmol/L. Both strategies are feasible. The outcomes after PAI in our center must be offset by much longer waitlist time due to the sensitization status of these patients. Data from multicentre experience will allow more robust comparative outcomes to be made, the current observations being restricted to a limited patient set.