R. Ramanathan1, G. Pettinato3, M. Mangino1, D. Lee4, X. Wen3, R. A. Fisher2 1Virginia Commonwealth University Medical Center,Surgery,Richmond, VA, USA 2Beth Israel Deaconess Medical Center,Transplantation,Boston, MA, USA 3Virginia Commonwealth University,Bioengineering,Richmond, VA, USA 4Mayo Clinic,Transplantation,Jacksonville, FL, USA
Introduction:
Hepatocyte cell transplantation can be life-saving in patients with acute liver failure (ALF), however it is limited by donor hepatocyte scarcity. We investigate the effect and function of stem cell-derived hepatocyte-like cells in an animal transplant model of ALF.
Methods:
A model of near-lethal ALF was developed in athymic nude rats using intraperitoneal D-galactosamine. A cell xeno-transplant model in rats was established using hepatic cells isolated from mouse livers. Human induced pluripotent stem cells (iPSC) and human mesenchymal stem cells (MSC) were differentiated into hepatocyte-like cells using a novel stem cell embryoid body suspension culture protocol. A subset of stem cell-derived hepatocyte-like cells were co-cultured with endothelial cells. Stem cell-derived hepatocyte-like cells were transplanted into the spleens of athymic nude rats with ALF with monitoring of survival and in vivo function through the presence of human albumin in the rat serum.
Results:
A reliable near-lethal model of acute liver failure was achieved with 975 mg/kg of D-galactosamine in rats weighing 270-350mg with >90% death within 3 days. Hepatic cells were reliably isolated from mouse livers with >85% viability and used to establish positive controls. In vitro differentiation of iPSC and MSC into hepatocyte-like cells was confirmed using genetic and protein studies. In vitro, stem cell-derived hepatocyte-like cells metabolized ammonia. They secreted albumin and fibrinogen in the presence of primary hepatic and endothelial cells respectively. 20 rats were transplanted with iPSC-derived hepatocyte-like cells and 12 rats were transplanted with MSC-derived hepatocyte-like cells. Compared to negative controls, transplant with iPSC-derived and MSC-derived hepatocyte-like cells were associated with increased survival (70% and 67% vs. 14%, p<0.05). Human albumin was detected in rat serum at three days after transplant in over half the animals transplanted with hepatocyte-like cells. Only animals transplanted with endothelial cell co-cultured hepatocyte-like cells had prolonged rat serum human albumin production. Despite transplantation into the spleen, human albumin was detected in the rat liver only, while human endothelial cells were detected in the rat spleen only (Figure).
Conclusion:
In a reliable near-lethal model of d-galactosamine induced acute liver failure, transplantation of stem cell-derived hepatocyte-like cells improved survival with evidence of in vivo cell functionality. Despite splenic transplantation, migration to cells into the liver is noted. Sustained in vivo function was noted in cells co-cultured with endothelial cells.
