ROLE OF THE CONSTITUTIVE ANDROSTANE RECEPTOR IN THE DERIVATION OF HEPATIC-LIKE CELLS FROM HUMAN EMBRYONIC STEM CELLS
Open Access
- Author:
- Zamule, Stephanie M.
- Graduate Program:
- Integrative Biosciences
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- March 22, 2010
- Committee Members:
- Curtis John Omiecinski, Dissertation Advisor/Co-Advisor
Curtis John Omiecinski, Committee Chair/Co-Chair
Gong Chen, Committee Member
Peter John Hudson, Committee Member
Gary H Perdew, Committee Member
John Patrick Vanden Heuvel, Committee Member
Kent Eugene Vrana, Committee Member - Keywords:
- embryonic stem cells
constitutive androstane receptor
liver
differentiation
nuclear receptor
development
lentivirus - Abstract:
- The liver performs an array of functions vital to life including detoxification, production of serum proteins, maintenance of cholesterol homeostasis, production and clearance of bile components, assembly and inter-conversion of amino acids, synthesis and breakdown of glucose, and processing of fatty acids. Current treatments for liver failure are inadequate, relying on liver or hepatocyte transplantation, both of which are significantly limited by insufficient donor tissue, donor-to-donor variability in tissue quality, and the risk of rejection, infection, or adverse immune response in the recipient. Human embryonic stem cells (hESCs) – derived from the inner cell mass of developing blastocysts and capable of giving rise to any cell type in the body upon exposure to the appropriate conditions – offer promise as an alternative source of cells from which a supply of hepatocytes may be derived for therapeutic transplantations. Hepatocytes derived from hESCs would also potentially provide a repository of cells for pharmacological and toxicological studies which rely on hepatocytes obtained from human donors as models for drug metabolism research and predictors of toxicological responses that may be associated with exposure to xenobiotic compounds. While a number of studies have demonstrated that hESCs are capable of differentiating into hepatic precursors, the precise means by which these cells may be derived, and the genes governing this multifaceted process, have yet to be fully elucidated. In this investigation we employed a unique hepatic differentiation protocol in which hESCs are cultured for only 10 days on collagen matrix in our hepatocyte media (William’s E Media supplemented with HEPES, glutamine, antibiotics, dexamethasone, insulin, transferrin, selenium, and linoleic acid/albumin). The resulting cell population exhibits hepatic-like cell morphology and decreased expression of ‘stemness’ markers including certain transcription factors, surface antigens, and enzymes. The hESC-derived hepatic-like cells express enhanced levels of hepatic markers including transcription factors, nuclear receptors, liver-generated plasma proteins, protease inhibitors, metabolic enzymes, and biotransformation enzymes. Acquisition of hepatic function is confirmed by the cells’ ability to transport anionic compounds and store glycogen. Notably, expression of the constitutive androstane receptor (CAR) – a nuclear receptor which, in the adult liver, is involved in the regulation of diverse physiological processes including all three phases of hepatic biotransformation and elimination as well as energy metabolism and lipid homeostasis – is highly increased in the hepatic-like cells, to levels approaching those of cultures of primary human hepatocytes. CAR is also expressed robustly and consistently in human fetal liver tissue obtained from subjects of a range of gestational ages. Modulation of CAR levels in differentiating hESCs using a lentivirus system – which we demonstrate to stably and robustly transduce both hESCs and cultures of primary human hepatocytes without affecting markers of pluripotency or the hepatic phenotype, respectively – reveals that CAR over-expression and siRNA-mediated attenuation of CAR mRNA levels result in corresponding changes in expression of hepatic markers previously assessed. In contrast, expression of pregnane X receptor (PXR) – the nuclear receptor exhibiting the most sequence homology to CAR – is not increased during hepatic differentiation and is negligible in human fetal liver tissue. Further, exogenous expression of PXR in hESCs induced to differentiate along a hepatic lineage does not result in enhanced expression of markers of the hepatic phenotype. Taken together, these results illuminate a unique, expeditious means by which functional hepatic-like cells may be derived from hESCs and further define a novel role for CAR in human hepatic specification.