The human body is constantly under assault by chemicals encountered in the environment.
Altering their molecular structure to increase polarity is essential for the elimination
from the body. Most bio-activation reactions are catalyzed by a group of hepatic microsomal
monooxygenases. Its reaction specificity is defined by cytochrome P450 (CYP), a family
of structurally related hemoproteins. Thus, CYPs are essential for the determination
of pharmacologic and toxic effects, which are conventionally measured in primary human
hepatocytes, whose availability is limited due to donor organ scarcity. This raises
the demand for alternatives. Hepatocyte-like (NeoHep) cells generated by trans-differentiation
of monocytes offer great perspectives.
NeoHep cells were prepared from peripheral blood monocytes. These cells were compared
to primary hepatocytes in terms of their morphology and metabolic behavior. CYP (1A2,
2A6, 2B6, 2C9, 2E1, and 3A4) expression and activity was investigated by RT-PCR, Westernblot,
and fluorescence-based microscale activity assays.
After 13–15 days of differentiation NeoHep cells form a confluent layer with cell-cell
contact displaying the typical hexagonal shape of primary hepatocytes, expressing
hepatocyte marker genes like transferrin and connexin 32. The basal CYP activity is
increasing throughout differentiation, reaching a stable level after approximately
15 days. Over a culture period of 5 days primary mouse hepatocytes undergo typical
morphologic changes in collagen monolayer culture and basal activity of all CYP isoforms
decreases. Responsiveness to inducers (e.g. 3-methylcholanthrene) and inhibitors (e.g.
Nifedipine) of CYP activity is comparable in both cell types.
Our data reveal phenotypic and metabolic similarities of NeoHep cells and primary
hepatocytes. Hence NeoHep cells might be used as an alternative for primary hepatocates
in measuring bioactivation of substances.
