Hepatocyte-Like Cells Can Be Derived from Human Umbilical Cord Blood and Embryonic Stem Cells: Tested in a Novel Mouse Model

Liver transplantation remains the only therapeutic option for many acute and chronic end-stage liver diseases. However, this approach is limited by a serious shortage of donor organs required for transplantation. Hepatocytes have been reported to be generated from cells not originated from liver, such as hematopoietic stem cells, mesenchymal stem cells and most recently embryonic stem cells. However, the frequency of these stem cell-derived hepatocytes is very low in most studies. Therefore, the significance of stem cell contribution to the repair of liver damage is still controversial. To further explore this potential, we used the beta-glucuronidase (GUSB)-null NOD/SCID/MPSVII mouse model for better identification of engrafted human cells. Enriched cord blood primitive cells (lineage depleted cells with high aldehyde dehydrogenase activity, ALDH^hiLin⁻) were transplanted into irradiated NOD/SCID/MPSVII mice. One month after transplantation, carbon tetrachloride (CCl₄) was administrated into the mice twice a week for 4 weeks to induce liver damage. In this model, ALDH^hiLin⁻ cells efficiently engrafted in the recipient mouse livers as demonstrated by GUSB positive immunohistological staining and the presence of human Alu DNA using PCR. The percentage of human cells in these livers ranged between 3% and 14.2% using quantitative real-time PCR. These engrafted cells improved recovery of the mice from toxic insult, and significantly increased the numbers of surviving mice. Furthermore, human liver-specific a-1-antitrypsin mRNA and albumin protein were expressed in the recipient livers. Interestingly, human vs. murine centromeric fluorescent in situ hybridization analysis on the liver sections demonstrated that most human cells were not fused to mouse cells. However, mouse nuclei were detected in the majority of the albumin-expressing cells, suggesting that fusion had occurred and was responsible for the appearance of donor derived hepatocyte-like cells. With the goal of achieving higher levels of liver reconstitution than had been possible using the adult stem cells, we began studying engraftment of human embryonic stem cells (hESC), which theoretically have the potential to regenerate any tissue. The H1 cell line was cultured on mouse embryonic fibroblasts then allowed to form embryoid bodies (EBs) in suspension culture for 7 days with or without further expansion and differentiation in attached culture for another month. EBs were dissociated into a single cell suspension and transplanted into NOD/SCID/MPSVII mice or NOD/SCID/IL2Rγ−/− mice via the tail vein after 300 RADs sublethal radiation with or without CCl₄ administration. Two months post-transplantation, the human EB-derived cells were found to be well engrafted in the NOD/SCID/MPSVII mouse livers, spleens and kidneys, using the clear-cut enzymatic identification method for cells expressing normal levels of beta-glucuronidase in the mice, which are null for the enzyme. Human DNA was also detected in the recipient mouse liver. Most interestingly, human albumin-expressing cells were also found in the livers of engrafted mice. Our data indicate that the progeny of cord blood stem cells can significantly enhance survival of mice with severe liver damage, and that fusion can occur between transplanted and recipient cells. This could be a normal mechanism of liver repair, since hepatocytes exist normally as multinucleate cells. We also demonstrate that the progeny of hESC can be effectively dissociated and transplanted intravenously, then home to the liver and differentiate to the hepatocyte lineage in an immune deficient mouse model of liver damage.