Paloma Maganto

Auxiliary liver by transplanted frozen-thawed hepatocytes.

Among the therapeutic alternatives to orthotopic liver transplantation, hepatocyte transplantation (HT) offers the best potential in a number of liver diseases, mainly inborn errors of metabolism. Nevertheless, HT presents several inconveniences such as the scarce knowledge of the functionality of the transplanted hepatocytes, which has given rise to controversy about the specificity or unspecificity of the transplant, and the lack of a suitable system for preserving the cells. This study was designed to test a system for cryopreserving hepatocytes and to assess their functionality over prolonged periods after their ectopic transplantation. A medium and a freezing schedule which are reproducible and yield elevated viability have been used, and a number of hepatospecific parameters have been assessed: the activity of ornithine carbamoyltransferase--an enzyme of primary importance in the urea cycle--lipogenesis, gluconeogenesis, glucose-6-phosphatase and cytochrome oxidase activities, the presence of albumin--as an index of plasma protein synthesis--and IDA uptake and metabolism, showing the UDP-glucuronyl transferase activity. As dedifferentiation markers, gamma-glutamyl transpeptidase and alpha-fetoprotein have been studied. From the results, it can be deduced that hepatocytes can be cryopreserved and transplanted and that under these conditions they maintain hepatic features for a long time. Following transplantation, several specific liver functions appear or are enhanced in the spleen. Freshly isolated and cryopreserved transplanted hepatocytes have similar behaviors, although a difference in the expression of the function can be observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Liver gene expression and increase in albumin synthesis by fetal hepatocytes transplanted into analbuminemic rats

This report describes the evolution of hepatocytes isolated from 21-day fetuses and transplanted into spleens of Nagase analbuminemic rats which have negligible serum albumin levels due to a mutation affecting albumin mRNA processing. Albumin and alpha-fetoprotein expression, in addition to other parameters related to cellular proliferation status (thymidine kinase and proliferating cell nuclear antigen expression) were studied as indicative of the behavior and evolution of the cells. In recipient rats, only a few clusters of hepatocytes could be observed in the red pulp of the spleen 24 h after transplantation. The fetal hepatocytes migrated to the liver and could be seen in portal branches immediately after transplantation. Fifteen days later, albumin mRNA was detected in recipient livers and was expressed throughout the entire 3-month study. Alpha-fetoprotein was not detected. Cell proliferation was not relevant, although 3 months after transplantation, the proliferation rates appeared to show a tendency to increase. These data demonstrate that fetal hepatocytes transplanted into spleen migrate to liver, settle there and acquire an adult phenotype free of malignant transformation. Our study is a first step towards the thorough understanding of fetal hepatocyte transplantation. The next steps will involve in-depth studies of the possibilities of genetic manipulation to achieve a high degree of repopulation/expression, employing the least possible number of donor cells, and of how the cells reach the liver parenchyma, overcoming the endothelial barrier.

Optimization of the technique to isolate fetal hepatocytes, and assessment of their functionality by transplantation.

In contrast to adult hepatocytes, fetal hepatocytes (FH) are thought to be highly proliferative less immunogenic and more resistant to both cryopreservation and ischemic injury. In the present study, we describe the method for isolation of FH and the relationship between the transplantability of FH into the spleen of analbuminemic rats and expression of albumin mRNA. Rat FH were obtained using the nonperfusion collagenase/DNase digestion method. Nagase analbuminemic rats (NAR), a strain which bears a mutation that determines the impossibility of the normal splicing of the albumin mRNA were used as recipients. The transplanted FH immediately migrated to the liver via portal vein, and anchored there. To assess the functional state of the transplanted cells, one month after transplantation, the expression of the albumin gene was studied in the liver of the recipients.

Survival of allogeneic hepatocytes transplanted into the thymus.

Background/Aim: Currently, when cell therapy is being considered instead of liver transplantation to treat terminal liver diseases, complete knowledge of the evolution and behavior of ectopically transplanted hepatocytes is a subject of utmost interest in the design of clinical trials. Hepatocytes survive in ectopic locations and have a therapeutic effect in different experimental models. Although it offers remarkable advantages over liver transplantation, hepatocyte transplantation presents several problems, among them the number of cells that can be injected at once and their rejection. Our main objective was to study the survival and functionality of hepatocytes transplanted into the thymus and, secondarily, to test whether the intrathymic transplant could induce any tolerogenic effect. Methods: Hepatocytes from F344 rats were transplanted into thymuses of Gunn rats, half of which received a unique dose of cyclosporine A. The recipients were sacrificed at different times. Light microscopy was performed and bilirubin levels were determined in serum and bile. Results/Conclusions: Transplanted hepatocytes survive for at least 6 weeks in the thymus of allogeneic animals without immunosuppressive therapy. The work provides interesting data about the behavior of hepatocytes injected into this unique ectopic site and shows that the thymus can be used as a recipient organ for cell therapy.

In Utero Hepatocellular Transplantation in Rats

This work represents a step forward in the experimental design of an in utero hepatocellular transplantation model in rats. We focused on the enrichment optimization of isolated fetal hepatocytes suspension, arranging the surgery methodology of in utero transplantation, monitoring the biodistribution of the transplanted hepatocytes, and assessing the success of the transplants. Rat fetuses have been transplanted at the 17th embryonic day (ED17) with fetal hepatocytes isolated from rats at the end of pregnancy (ED21). We assessed possible differences between lymphocyte population, CD4 positive, CD8 positive, double-positive T-cells, and anti-inflammatory cytokines interleukins 4 and 10 (IL4 and IL10) as well. Cellular viability reached the rates of 90–95%. Transplanted groups had a limited success. Transplanted hepatocytes were not able to pass through the hematoplacental barrier. The hepatocytes injected were primarily located in the liver. There was an upward trend in the whole amount of T CD4 and T CD8 cells. There was an increased IL4 in the transplanted groups observed in the pregnant rats. The possibility to induce tolerance in fetuses with a hepatocyte transplant in utero could be a key point to avoid the immunosuppression treatments which must be undergone by transplanted patients.