The treatment of inherited metabolic liver diseases by hepatocyte transplantation (HT) would be greatly facilitated if the transplanted normal hepatocytes could be induced to proliferate preferentially over the host liver cells. We hypothesized that preparative hepatic irradiation ( H epatocyte transplantation (HT) is currently being evaluated as a treatment strategy for patients with acute and chronic liver failure and to replace metabolic liver functions in inherited liver diseases. 1 HT has been used in the treatment of inherited metabolic diseases, such as Crigler-Najjar syndrome type I, 2 and for hepatocyte-based ex vivo gene therapy in experimental animals 3-5 as well as in patients with low-density lipoprotein receptor deficiency. 6 However, the clinical application of HT is limited by the availability of human hepatocytes and the number of liver cells that can be transplanted safely at one time. An important consideration is whether a sufficient number of hepatocytes can be engrafted to achieve the desired metabolic correction without causing portal hypertension or other adverse effects. Therefore, a method to induce preferential proliferation of a relatively small number of engrafted hepatocytes in vivo could markedly enhance the applicability of HT.We hypothesized that preparative irradiation of the liver along with a strong mitotic stimulus provided by a maneuver such as partial hepatectomy (PH) should damage the host hepatocyte DNA, causing cell cycle arrest. Subsequently transplanted normal, nonirradiated hepatocytes should proliferate preferentially in response to the

Over the years, substantial evidence has accumulated suggesting the existence of potential liver stem cells (LSCs) in the adult liver. In all cases, the putative LSCs were activated to proliferate and differentiate when the regenerative capacity of terminally differentiated hepatocytes was compromised. The progeny of potential LSCs, referred to as oval cells, behave like bipotential progenitors capable of differentiation into mature hepatocytes and biliary epithelial cells, thus recapitulating hepatoblast differentiation during fetal development [1][2][3][4][5]. Oval cells also reveal some phenotypic characteristics of hematopoietic progenitor cells; they express c-kit and its ligand stem cell factor [6] and the related flt-3 and flt-3 ligand [7]

Flt3 ligand (Flt3L) therapy that expands dendritic cells in vivo in combination with local tumor radiotherapy (RT) significantly improved survival and induced a long-term tumor-specific immune response in a murine model of Lewis lung carcinoma (3LL). The irradiated tumor cells were able to significantly restimulate the splenocytes of the RT + Flt3L cohort in vitro. The restimulated splenocytes demonstrated increased cytotoxic response, lymphocytic proliferation and elevated levels of Th type I cytokines (IL-2, IL-12, IFN-γ and TNF-α). The combination therapy of RT + Flt3L induced a long-term protective immunity in the disease-free animals. The protective effect was further enhanced when the disease-free animals were vaccinated with irradiated tumor cells. The vaccinated animals had significantly greater protection compared to the nonvaccinated group against subsequent challenge with 3LL cells. Taken together, these results indicate that the release of tumor antigens by irradiated dying tumors and concomitant administration of Flt3L was able to facilitate the generation of a tumor-specific long-term immune response against a poorly immunogenic tumor. This effect was further boosted by vaccination with irradiated tumor cells.

A strategy for inducing preferential proliferation of the engrafted hepatocytes over host liver cells should markedly increase the benefit of hepatocyte transplantation for the treatment of liver diseases and ex vivo gene therapy. We hypothesized that preparative hepatic irradiation (HIR) to inhibit host hepatocellular regeneration in combination with the mitotic stimulus of host hepatocellular apoptosis should permit repopulation of the liver by transplanted cells. To test this hypothesis, congeneic normal rat hepatocytes were transplanted into UDP-glucuronosyltransferase (UGT1A1)-deficient jaundiced Gunn rats (a model of Crigler-Najjar syndrome type I), following HIR and adenovirus-mediated FasL gene transfer. Progressive repopulation of the liver by engrafted UGT1A1-proficient hepatocytes over 5 months was demonstrated by the appearance of UGT1A1 protein and enzyme activity in the liver, biliary bilirubin glucuronides secretion, and long-term normalization of serum bilirubin levels. This is the first demonstration of massive hepatic repopulation by transplanted cells by HIR and FasL-induced controlled apoptosis of host liver cells.

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Scarcity of donor livers is a major obstacle to the general application of hepatocytes for the development of bioartificial liver assist devices as well as intracorporeal engraftment of hepatocytes for the treatment of inherited metabolic diseases. The number of hepatocytes that can be transplanted into the liver safely in a single sitting also limits the utility of this procedure. These limitations could be addressed by providing preferential proliferative advantage to the transplanted cells. Studies using transgenic mouse recipients or donors have indicated that massive repopulation of the host liver by engrafted hepatocytes requires that the transplanted cells are subjected to a proliferative stimulus to which the host hepatocytes cannot respond. Prevention of host hepatocyte proliferation has been achieved by treatment with a plant alkaloid, retrorsine. Because retrorsine is carcinogenic, we have evaluated preparative irradiation for this purpose. The proliferative stimulus may consist of the loss of hepatic mass (e.g., partial hepatectomy, reperfusion injury or induction of Fas-mediated apoptosis by gene transfer) or administration of stimulants of hepatocellular mitosis (e.g., growth factors or thyroid hormone). Potential applications of these preparative manipulations of the host liver include the treatment of inherited metabolic disorders by transplantation of allogeneic hepatocytes, hepatocyte-mediated ex vivo gene therapy, rescuing liver cancer patients from radiation-induced liver damage, and expansion of human hepatocytes in animal livers.

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