Hongbo Cai

Stem cell characteristics of amniotic epithelial cells.

Amniotic epithelial cells develop from the epiblast by 8 days after fertilization and before gastrulation, opening the possibility that they might maintain the plasticity of pregastrulation embryo cells. Here we show that amniotic epithelial cells isolated from human term placenta express surface markers normally present on embryonic stem and germ cells. In addition, amniotic epithelial cells express the pluripotent stem cell–specific transcription factors octamer‐binding protein 4 (Oct‐4) and nanog. Under certain culture conditions, amniotic epithelial cells form spheroid structures that retain stem cell characteristics. Amniotic epithelial cells do not require other cell‐derived feeder layers to maintain Oct‐4 expression, do not express telomerase, and are nontumorigenic upon transplantation. Based on immunohistochemical and genetic analysis, amniotic epithelial cells have the potential to differentiate to all three germ layers—endoderm (liver, pancreas), mesoderm (cardiomyocyte), and ectoderm (neural cells) in vitro. Amnion derived from term placenta after live birth may be a useful and noncontroversial source of stem cells for cell transplantation and regenerative medicine.

Disrupted Bile Acid Homeostasis Reveals an Unexpected Interaction among Nuclear Hormone Receptors, Transporters, and Cytochrome P450

Sister of P-glycoprotein (SPGP) is the major hepatic bile salt export pump (BSEP). BSEP/SPGP expression varies dramatically among human livers. The potency and hierarchy of bile acids as ligands for the farnesyl/bile acid receptor (FXR/BAR) paralleled their ability to induce BSEP in human hepatocyte cultures. FXR:RXR heterodimers bound to IR1 elements and enhanced bile acid transcriptional activation of the mouse and human BSEP/SPGP promoters. In FXR/BAR nullizygous mice, which have dramatically reduced BSEP/SPGP levels, hepatic CYP3A11 and CYP2B10 were strongly but unexpectedly induced. Notably, the rank order of bile acids as CYP3A4 inducers and activators of pregnane X receptor/steroid and xenobiotic receptor (PXR/SXR) closely paralleled each other but was markedly different from their hierarchy and potency as inducers of BSEP in human hepatocytes. Moreover, the hepatoprotective bile acid ursodeoxycholic acid, which reverses hydrophobic bile acid hepatotoxicity, activates PXR and efficaciously induces CYP3A4 (a bile-metabolizing enzyme) in primary human hepatocytes thus providing one mechanism for its hepatoprotection. Because serum and urinary bile acids increased in FXR/BAR −/− mice, we evaluated hepatic transporters for compensatory changes that might circumvent the profound decrease in BSEP/SPGP. We found weak MRP3 up-regulation. In contrast, MRP4 was substantially increased in the FXR/BAR nullizygous mice and was further elevated by cholic acid. Thus, enhanced hepatocellular concentrations of bile acids, due to the down-regulation of BSEP/SPGP-mediated efflux in FXR nullizygous mice, result in an alternate but apparent compensatory up-regulation of CYP3A, CYP2B, and some ABC transporters that is consistent with activation of PXR/SXR by bile acids.

Hepatocyte Transplantation: Clinical Experience and Potential for Future Use

Hepatocyte transplantation has been proposed as a method to support patients with liver insufficiency. There are three main areas where the transplantation of isolated hepatocytes has been proposed and used for clinical therapy. Cell transplantation has been used: 1) for temporary metabolic support of patients in end-stage liver failure awaiting whole organ transplantation, 2) as a method to support liver function and facilitate regeneration of the native liver in cases of fulminant hepatic failure, and 3) in a manner similar to gene therapy, as a “cellular therapy” for patients with genetic defects in vital liver functions. We will briefly review the basic research that leads to clinical hepatocyte transplantation, the published clinical experience with this experimental technique, and some possible future uses of hepatocyte transplantation.

INDUCTION OF CYP3A IN PRIMARY CULTURES OF HUMAN HEPATOCYTES BY GINKGOLIDES A AND B

1 The present study was designed to determine the effects of ginkgolide A, ginkgolide B and quercetin on CYP3A protein expression and enzyme activity in primary cultures of human hepatocytes. 2 Hepatocytes were pretreated with ginkgolide A, ginkgolide B and quercetin (at 1, 3, 10 and 30 µmol/L) for 48 h and then exposed to testosterone (250 µmol/L) for 30 min. Rifampin (10 µmol/L) and phenobarbital (2 mmol/L) were used as positive controls. The CYP3A activity was measured by the amount of 6β‐hydroxytestosterone in the culture medium and CYP3A protein in hepatocyte lysate was semiquantified by immunoblotting. 3 Compared with the vehicle control, ginkgolides A and B, at 30 µmol/L, significantly induced CYP3A protein expression (2.1‐ and 2‐fold, respectively; both P < 0.01) and markedly induced CYP3A‐mediated testosterone 6β‐hydroxylation (2.5‐fold each; P < 0.05 for ginkgolide A; P > 0.05 for ginkgolide B). Quercetin had no apparent induction. 4 Ginkgolide A and ginkgolide B can induce CYP3A protein expression and enzyme activity in primary cultures of human hepatocytes at higher doses.

Bigger may not be better when it comes to hepatocytes

Hepatocyte transplantation has gained attention as a potential therapeutic intervention for a number of acute and chronic diseases including fulminant hepatic failure, metabolic liver disease, and cirrhosis. Several decades of research with small animal models has shown that hepatocyte transplants can support liver function and life in animals in acute liver failure. Animal models of metabolic liver disease including tyrosinemia type 1, Crigler-Najjar (Gunn rat), analbuminemia, urea cycle disorders, primary familial intrahepatic cholestasis (PFIC), and Wilson’s disease are among the many disorders that have been at least partially corrected by hepatocyte transplantation. A number of centers around the world have conducted hepatocyte transplants in patients in liver failure or even more frequently in patients with metabolic defects in liver function. The results in the clinics are in general agreement with the preclinical data with animal models and support the hypothesis that continued efforts in this field will help bring this cellular therapy to more general use. One of the problems with hepatocyte transplants is the availability of cells for transplantation. The normal sources of hepatocytes are donor livers that cannot be used for whole organ transplantation. This requires that transplant quality cells be isolated from organs that were rejected for whole organ transplant. New sources for hepatocytes will be needed to expand the use of hepatocyte transplants to additional medical centers. Suggested cell sources include immortalized human liver lines, xenotransplants, and stem cell derived sources. A report in this issue by Shibata et al., (this issue) suggests that populations of small hepatocytes (SH) derived over a 12-day period from cultured rat hepatocytes may be useful for cell transplantation. Following transplantation of fresh hepatocytes or SH, liver repopulation was nearly identical. However, according to the author’s calculations at least 10-fold fewer SH were transplanted as compared to freshly isolated total hepatocytes, suggesting that on a cell by cell basis SH may engraft or proliferate better following transplantation than the general population of cells isolated from adult liver. Small hepatocytes, a term used to describe the morphology of the proliferating colonies of cells that are observed when rat hepatocytes are cultured in media supplemented with nicotinamide and growth factors such as EGF, were first described in 1992 by Mitaka et al. Characteristics of SH cells put them somewhere between fully mature hepatocytes and progenitor cells such as oval cells in that SH express some adult liver functions such as albumin, transferrin, and cytokeratins 8 and 18 but express little to no alpha-fetoprotein, CK14, OC2, or the placental type of glutathione S-transferase as would be expected of a progenitor cell. Similar experiments conducted by other investigators confirm these observations. Importantly, SH display a high proliferative rate in culture with up to 86% of the cells labeled with BrdU out to 8 days of culture. Proliferation and maturation of liver functions of SH in this culture system is accompanied by the proliferation of non-parenchymal cells from the liver including epithelial, sinusoidal endothelial, and stellate or Ito cells that initially surround the colonies and later invade under the colonies and support their continued maturation. Similar observations were made by Michalopoulos et al.