Hepatocyte transplantation has been proposed as a method to support patients with liver insufficiency. There are three main areas where the transplantation of isolated hepatocytes has been proposed and used for clinical therapy. Cell transplantation has been used: 1) for temporary metabolic support of patients in end-stage liver failure awaiting whole organ transplantation, 2) as a method to support liver function and facilitate regeneration of the native liver in cases of fulminant hepatic failure, and 3) in a manner similar to gene therapy, as a “cellular therapy” for patients with genetic defects in vital liver functions. We will briefly review the basic research that leads to clinical hepatocyte transplantation, the published clinical experience with this experimental technique, and some possible future uses of hepatocyte transplantation.
Isolation and Characterization of Mesenchymal Stem Cells from Human Fetal Liver; Potential Candidates for Replacement Therapy in Liver Disease Liver cell transplantation (LCT) is emerging as an alternative to organ transplantation in the treatment of liver disease since tissue unavailability and immunological incompatibilities are still major obstacles. Primary human hepatocytes are the first choice for LCT. However the unavailability of liver donors, the need for high numbers of hepatocytes for transplantation, immunological rejection, and low activity and inability to expand hepatocytes in vitro are limiting factors.
Transplantation of human hepatocytes (HTx) has gained recognition as a bridge to, or an alternative to, orthotopic liver transplantation for patients with acute liver failure or genetic defects in liver function. Although the quality of the hepatocytes used for cell transplantation is critical, no consensus exists on protocols to assess the function of hepatocytes prior to HTx. Application of this cell therapy in clinical practice could be aided by fast and reliable assays to evaluate the functional competence of isolated hepatocytes prior to clinical transplantation. Traditional assays for measuring metabolic functions in primary hepatocytes frequently involve highly technical equipment, time-consuming methods, and large numbers of cells. We describe a novel approach for the rapid assessment of the metabolic capabilities of human hepatocytes. This report details simple procedures to evaluate 11 endpoints from cells isolated from human liver that can be performed by a single operator within approximately 2 h of isolation. Longer term cultured hepatocytes were also analyzed to determine if the results from the 2-h tests were predictive of long-term hepatic function. The assays simultaneously measure five cytochrome P450 activities, one phase II activity, plating efficiency, and ammonia metabolism in addition to viability and cell yield. The assays require fewer than 20 million cells and can be completed using commonly available and inexpensive laboratory equipment. The protocol details methods that can be used in a time frame that would allow analysis of hepatic functions in freshly isolated hepatocytes prior to their use for clinical transplantation.
The regulation of hepatic bile acid formation is incompletely understood. Primary cultures of mammalian hepatocytes offer an opportunity to examine putative regulatory factors in relative isolation. Using rat and human hepatocytes in primary culture, we examined bile acid composition and the expression of the rate-limiting enzyme of formation, cholesterol 7α-hydroxylase. Control rat hepatocytes showed a declining bile acid production over 4 days, from 156 ± 24 ng/mL (67% cholic acid) on day 1 to 55 ± 11 ng/mL (55% cholic acid) on day 4. In addition to cholic acid, chenodeoxycholic acid, α-muricholic acid, and β-muricholic acid were formed. Treatment with triidothyronine (T 3 ) or dexamethasone alone had no significant effect on bile acid production. A combination of T 3 and dexamethasone significantly increased the total bile acid production on day 4 (224 ± 54 ng/mL) and resulted in a marked change in composition to 23% cholic acid and 77% non-12α-hydroxylated bile acids. Control rat hepatocytes had a cholesterol 7α-hydroxylase activity of 3.3 ± 0.6 pmol/mg protein/min after 4 days in culture. Cells treated with the combination of dexamethasone and T 3 had an activity of 16.4 ± 3.6 pmol/mg protein/min. The cholesterol 7α-hydroxylase messenger RNA (mRNA) levels, determined by solution hybridization after 4 days of culture, showed results similar to those for the activity data; control cells had 5.3 ± 0.9 cpm/μg total nucleic acids (tNAs). T 3- or dexamethasone-treated cells did not differ from control cells, whereas the combination of T 3 and dexamethasone increased the mRNA levels to 20.6 ± 2.8 cpm/μg tNAs. In human hepatocytes, isolated from donor liver, bile acid formation increased from 206 ± 79 ng/mL on day 2 to 1490 ± 594 ng/mL on day 6 and then declined slightly. Cholic acid and chenodeoxycholic acid were formed, constituting about 80% and 20%, respectively. The combined addition of T 3 and dexamethasone had a tendency to decrease rather than increase bile acid formation. Also, mRNA levels of the cholesterol 7α-hydroxylase increased severalfold in the human hepatocytes from day 2 to day 4 and then declined. The addition of T 3 or dexamethasone did not effect the mRNA levels in any consistent way. It is noteworthy that the capacity of the cultured human hepatocytes to produce bile acids was higher than that of cultured rat hepatocytes, in spite of the fact that the production of bile acids in rat liver is 3- to 5-fold higher than that in human liver in vivo . It is also evident that while hormonal factors appear to regulate bile acid synthesis in the rat, no evidence for this was found in human hepatocytes. As the composition of bile acids secreted by human hepatocytes in primary culture closely resembles that found in vivo , this represents a useful model for further studies of the synthesis and regulation of bile acids.
Journal Article Teacher's Section Get access Elmer Ellis Elmer Ellis Editor Search for other works by this author on: Oxford Academic Google Scholar Journal of American History, Volume 26, Issue 1, June 1939, Pages 71–73, https://doi.org/10.1093/jahist/26.1.71 Published: 01 June 1939
On September 6 and 7, 2009 a meeting was held in London to identify and discuss what are perceived to be current roadblocks to effective hepatocyte transplantation as it is currently practiced in the clinics and, where possible, to offer suggestions to overcome the blocks and improve the outcomes for this cellular therapy. Present were representatives of most of the active clinical hepatocyte transplant programs along with other scientists who have contributed substantial basic research to this field. Over the 2-day sessions based on the experience of the participants, numerous roadblocks or challenges were identified, including the source of cells for the transplants and problems with tracking cells following transplantation. Much of the discussion was focused on methods to improve engraftment and proliferation of donor cells posttransplantation. The group concluded that, for now, parenchymal hepatocytes isolated from donor livers remain the best cell source for transplantation. It was reported that investigations with other cell sources, including stem cells, were at the preclinical and early clinical stages. Numerous methods to modulate the immune reaction and vascular changes that accompany hepatocyte transplantation were proposed. It was agreed that, to obtain sufficient levels of repopulation of liver with donor cells in patients with metabolic liver disease, some form of liver preconditioning would likely be required to enhance the engraftment and/or proliferation of donor cells. It was reported that clinical protocols for preconditioning by hepatic irradiation, portal vein embolization, and surgical resection had been developed and that clinical studies using these protocols would be initiated in the near future. Participants concluded that sharing information between the groups, including standard information concerning the quality and function of the transplanted cells prior to transplantation, clinical information on outcomes, and standard preconditioning protocols, would help move the field forward and was encouraged.
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