The second population contains hepatocytes coexpressing one or two growth-regulatory molecules (growth factor or Erlotinib oncogene) plus a human placental alkaline phosphatase (hPAP) marker transgene.14 Recipient animals express the major urinary protein uPA (urokinase-type plasminogen activator) transgene, which is hepatotoxic.14 The transient liver disease that is present in these mice provides a growth-stimulatory environment for transplanted donor hepatocytes, and the donor cells proliferate and expand as clonal foci for approximately 4 weeks. By this time, diseased hepatic parenchyma has

been replaced by a combination of healthy donor hepatocytes and healthy endogenous hepatocytes that have inactivated uPA transgene expression, and the repopulated liver becomes quiescent.14, 16 Donor cell number was chosen so that donor repopulation of liver would be minimal (<5%),14 thereby enabling accurate measurement of donor focus size uncomplicated by the presence of multiple adjoining foci. We compare the size of foci from the two donor cell populations at 1, 2, 4, 8, and 12 weeks posttransplantation,

using histochemistry to distinguish between foci expressing selleck chemicals llc β-galactosidase (the lacZ gene product) or hPAP. For each oncogene evaluated, we use at least two donor cell preparations, and multiple recipients are examined at each time posttransplantation. We also performed histochemical, immunohistochemical, or in situ RNA hybridization assays to detect donor cell transgene expression in representative recipient animals. Coexpression of all transgenes was identified in 90%-99% of foci with each transgene combination (Table 1). In control experiments, we measured mean areas of transplant foci in recipients receiving normal hPAP (with no oncogenic transgene) and normal Hormones antagonist lacZ donor cells (Fig. 2A). We observed no significant differences in focus size except at 1

week posttransplantation. The difference at 1 week is likely a measurement artifact caused by the nuclear localization of β-gal versus the cell surface and cytoplasmic localization of hPAP. In larger (older) foci, this does not bias measured focus size. Thus, markers do not differentially affect focus growth. We identified two growth stages of normal donor cells in recipient livers (Fig. 2A). First was a “growth phase,” from weeks 1 to 4 posttransplantation, during which donor hepatocyte foci increase in size. Second was a “quiescence phase,” from weeks 4 to 12 posttransplantation, which is related to completion of parenchymal repopulation by healthy cells and corresponding loss of the growth stimulus.14, 16 During this stage, control donor focus size remains constant. We also calculated the number of hPAP-marked donor hepatocyte cell doublings required to generate the observed median focus sizes at 4 and 12 weeks posttransplantation (Table 2; for this and subsequent analyses we evaluate median data).