2013 - CTS 2013 Congress

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Oral Communications 11

23.1 - Characterization of Metabolic Activities from Isolated Fetal Human Hepatocytes

Presenter: Roberto, Gramignoli, Stockholm, Sweden
Authors: Roberto Gramignoli1,2, Veysel Tahan1,2, Kenneth Dorko1,2, Kristen Skvorak1,2, Stephen Strom1,2

Characterization of Metabolic Activities from Isolated Fetal Human Hepatocytes

Roberto Gramignoli1,2, Veysel Tahan1,2, Kenneth Dorko1,2, Kristen Skvorak1,2, Stephen Strom1,2

1Dept. of Pathology, Karolinska University Hospital, Stockholm, Sweden; 2Dept. of Pathology, University of Pittsburgh, Pittsburgh, PA, United States


Introduction:Hepatocyte transplantation (HTx) is an alternative to liver transplantation for certain liver diseases. Clinical HTx programs are hampered by the shortage of hepatocytes for this therapy. Fetal hepatocytes have been proposed as an alternative source of cells for transplants, however, little information is available concerning the metabolic activities of fetal cells as compared to adults.  Aim: The viability and metabolic activity of fetal hepatocytes isolated from donors of different gestational ages were assessed and compared to the values obtained from pediatric and adult hepatocytes.
Methods: Hepatocytes were isolated from 252 fetal liver tissues of 11-24 weeks gestational age and 200 “adult” hepatocyte preparations (age range 3 months-85 years).  Cell viability, recovery and apoptosis were evaluated immediately post-isolation. When possible, data on 11 different measurements of hepatocyte function including ATP content, plating efficiency basal and induced Cytochrome P450 (CYP) activities, phase II conjugation and ammonia metabolism were collected and analyzed.
Results:Cells isolated from fetal tissues had a significant higher viability compared to adult hepatocytes (91+7% vs 78+12%, mean+SD; p<0.0001), and a 2-fold higher level of apoptosis. Specific hepatic functions were measured on approximately 2/3rds of the preparations. ATP content was significantly higher in fetal hepatocytes compared to adult cell preparations (328+123 vs 32+38 LCU/min/103 hepatocytes; p<0.0001). Up to 500-fold differences were observed between different gestational and post-natal ages in functional assays. In fetal hepatocytes, a robust induction (up to 30-fold) of CYP enzymes was observed after three days of exposure to specific CYP inducers. The fetal form of CYP3A (3A7) was expressed at low level at early gestational ages, and its activity increased during fetal development, reaching the maximum value in hepatocytes isolated from postnatal infant tissues (<3 years in age). A continuous increase was observed in CYP3A4 and 1A activity from fetal to adult donors. Phase II metabolism, measured as the conjugation of Resorufin by hepatocytes, was easily measured in fetal cells immediately after isolation; however, this activity did not increase in culture as is usually observed in mature hepatocytes. Ammonia metabolism was null or low immediately after isolation in fetal hepatocytes, but increased to approximately adult levels if cells were cultured for 3-5 days (123+101 nmol/min/mg). 
Conclusions: Viability and metabolic functions were measured from almost 500 hepatocyte preparations, from fetal, infant and adult human liver tissues.  Data is presented on the normal ranges for distinct hepatic functions from attachment to ATP content and ammonia metabolism, to phase I and II drug metabolism in each age group.  The inducibility of CYP enzymes and the rapid maturation of ammonia metabolism suggest that fetal hepatocytes could be useful for clinical hepatocyte transplantation.

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