Upregulation of CYP 450s expression of immortalized hepatocyte-like cells derived from mesenchymal stem cells by enzyme inducers

<p>Abstract</p> <p>Background</p> <p>The strenuous procurement of cultured human hepatocytes and their short lives have constrained the cell culture model of cytochrome P450 (CYP450) induction, xenobiotic biotransformation, and hepatotoxicity. The development of continuous non-tumorous cell line steadily containing hepatocyte phenotypes would substitute the primary hepatocytes for these studies.</p> <p>Results</p> <p>The hepatocyte-like cells have been developed from hTERT plus Bmi-1-immortalized human mesenchymal stem cells to substitute the primary hepatocytes. The hepatocyte-like cells had polygonal morphology and steadily produced albumin, glycogen, urea and UGT1A1 beyond 6 months while maintaining proliferative capacity. Although these hepatocyte-like cells had low basal expression of CYP450 isotypes, their expressions could be extensively up regulated to 80 folds upon the exposure to enzyme inducers. Their inducibility outperformed the classical HepG2 cells.</p> <p>Conclusion</p> <p>The hepatocyte-like cells contained the markers of hepatocytes including CYP450 isotypes. The high inducibility of CYP450 transcripts could serve as a sensitive model for profiling xenobiotic-induced expression of CYP450.</p

The molecular basis of beta-thalassemia intermedia in southern China: genotypic heterogeneity and phenotypic diversity

<p>Abstract</p> <p>Background</p> <p>The clinical syndrome of thalassemia intermedia (TI) results from the β-globin genotypes in combination with factors to produce fetal haemoglobin (HbF) and/or co-inheritance of α-thalassemia. However, very little is currently known of the molecular basis of Chinese TI patients.</p> <p>Methods</p> <p>We systematically analyzed and characterized β-globin genotypes, α-thalassemia determinants, and known primary genetic modifiers linked to the production of HbF and the aggravation of α/β imbalance in 117 Chinese TI patients. Genotype-phenotype correlations were analyzed based on retrospective clinical observations.</p> <p>Results</p> <p>A total of 117 TI patients were divided into two major groups, namely heterozygous β-thalassemia (n = 20) in which 14 were characterized as having a mild TI with the Hb levels of 68-95 g/L except for five co-inherited ααα^anti-3.7triplication and one carried a dominant mutation; and β-thalassemia homozygotes or compound heterozygotes for β-thalassemia and other β-globin defects in which the β⁺-thalassemia mutation was the most common (49/97), hemoglobin E (HbE) variants was second (27/97), and deletional hereditary persistence of fetal hemoglobin (HPFH) or δβ-thalassemia was third (11/97). Two novel mutations, Term CD+32(A→C) and Cap+39(C→T), have been detected.</p> <p>Conclusions</p> <p>Chinese TI patients showed considerable heterogeneity, both phenotypically and genotypically. The clinical outcomes of our TI patients were mostly explained by the genotypes linked to the β- and α-globin gene cluster. However, for a group of 14 patients (13 β⁰/β^Nand 1 β⁺/β^N) with known heterozygous mutations of β-thalassemia and three with homozygous β-thalassemia (β⁰/β⁰), the existence of other causative genetic determinants is remaining to be molecularly defined.</p

Investigation of FoxO3 dynamics during erythroblast development in β-thalassemia major

<div><p>The FoxO3 transcription factor is a key regulator of oxidative stress and erythroid maturation during erythropoiesis. In this study, we explored the involvement of FoxO3 in severe β-thalassemia. Using primary CD34⁺ hematopoietic progenitor cells from patients with β-thalassemia major, we successfully developed an <i>in vitro</i> model of ineffective erythropoiesis. Based on this model, FoxO3 activity was quantified in single cells using high throughput imaging flow cytometry. This study revealed a significant reduction of FoxO3 activity during the late stage of erythroblast differentiation in β-thalassemia, in contrast to erythropoiesis in normal cells that maintain persistent activation of FoxO3. In agreement with the decreased FoxO3 activity in β-thalassemia, the expression of FoxO3 target genes was also found to decrease, concurrent with elevated phosphorylation of AKT, most clearly at the late stage of erythroid differentiation. Our findings provide further evidence for the involvement of FoxO3 during terminal erythropoiesis and confirm the modulation of the PI3K/AKT pathway as a potential therapeutic strategy for β-thalassemia.</p></div

Clinical characteristics of patients.

<p>Clinical characteristics of patients.</p

Decreased FoxO3 nuclear localization during late erythroblast maturation in β-thalassemia.

<p>Imaging flow cytometry analysis with FoxO3/nucleus similarity scores of each stage of erythroblasts from normal and β-thalassemia samples on day 11 (A) and day 14 (B). (C) Analysis of similarity scores of OrthoE in normal and β-thalassemia on day 14 with representative brightfield (BF) images, fluorescence images, and similarity scores (white numbers). Summary of % FoxO3 nuclear translocation in each stage of normal and β-thalassemia erythroblasts on day 11 (D) and day 14 (E). The data from normal cells on day 11 presented in Figs 3A and D were based on those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187610#pone.0187610.g002" target="_blank">Fig 2D</a>. Data are expressed as means ± SEM. **<i>P</i> < 0.01 was calculated using the Student’s t test. Normal group, n = 5 independent experiments (from 5 different normal subjects); β-thalassemia group, n = 4 independent experiments (from 4 different patients).</p

β-Thalassemia cells shows higher levels of phosphorylated AKT and FoxO3 and exhibit lower expression of FoxO3 target genes in late erythroid differentiation.

<p>(A) Expression of pAKT, total AKT, pFoxO3, and total FoxO3 in cultured cells on day 14 from normal and β-thalassemia groups analyzed by immunoblotting. β-Actin used as a protein loading control. Quantification of relative pAKT and pFoxO3 levels (Right panel). The data are presented as pAKT/AKT or pFoxO3/FoxO3 ratios and shown as means ± SEM. *<i>P</i> < 0.05, n = 3 independent experiments. (B) Relative mRNA expression levels of <i>CAT</i>, <i>SOD2</i>, <i>BIM</i>, <i>RIOK3</i>, <i>PINK1</i>, and <i>ULK1</i> in normal and β-thalassemia on day 11 and day 14 evaluated by real-time RT-PCR. <i>GAPDH</i> used as a reference gene. Data are expressed as means ± SEM. *<i>P</i> < 0.05, **<i>P</i> < 0.01 by Student’s t test.</p