Identification of biopotent hepatic stem cells, able to regenerate both hepatocytes and cholangioctyes, could be of key therapeutic importance in the development of artificial livers, techniques of liver cell transplantation, and for use as target cells in gene therapies. Recent work indicates that the bone marrow of rodents harbors hepatic stem cells. The short-term goal of this project is to determine the kinetics of hepatic engraftment of bone marrow cells in mice by stimulating hepatic engraftment via different models of hepatic injury. CD34+lin- male, beta-galactosidase positive marrow cells, previously shown to accomplish such engraftment, will be transplanted into female, beta-galactosidase negative mice. Fluorescence in situ hybridization for Y-chromosome and histochemical staining for cytoplasmic bete-galactosidase will identify hepatocytes and cholangiocytes of bone marrow origin. Hepatic engraftment of marrow cells will be quantitatively and topographically evaluated in different models of hepatic regeneration including: without any source of hepatic injury (c-kit deficient mutant mice); requiring no radiation, low dose radiation or myeloablative radiation; well characterized toxic injuries of specific cell compartments (perivenular and periportal hepatocytes, biliary epithelium); and pan-organ hypertrophy after partial hepatectomy. The long term aim is to determine if this hepatic engraftment is functional. The most reproducible models from the first phase of work will be applied to correction of genetic defects in hepatocytes, in particular, hepatocyte apical and basolateral secretory defects. Specifically, correction of hepatocyte secretory defects will be tested by transplanting: female transgenic mice expressive of human abnormal (PiZ) a-1-antitrypsin (A1AT) with marrow from male mice expressive of human normal (PiM) A1AT (basolateral defect); female mdr2 knockout mice, mimicking human Progressive Familial Intrahepatic Cholestasis, with bone marrow from male transgenic mice expressive of human MDR3 (apical defect). Finally, evidence of hepatic engraftment of marrow cells in humans will be sought by examining clinical (biopsy and autopsy) specimens from female recipients of therapeutic bone marrow transplants from male donors to determine applicability of these studies for treatment of human diseases.
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