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

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

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

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

<i>In vitro</i> differentiation of CD34⁺ cells towards erythrocytes recapitulates ineffective erythropoiesis seen in β-thalassemia.

<p>(A) Imaging flow cytometry gating strategy for identifying each distinct stage of erythroblast populations. (B) Morphology of each stage of erythroblasts analyzed by imaging flow cytometry (merged fluorescence images of CD36 (blue) and propidium iodide (orange) representing cytoplasm and nucleus areas respectively), compared with the morphology observed in Giemsa-stained cytospin preparations (brightfield images). (C) Immunofluorescence images of erythroid cell surface markers CD36 (blue), CD71 (red) and GlyA (pink) in each stage of erythroblasts captured using an imaging flow cytometer. (D) Percentage of each stage of erythroblasts in normal and β-thalassemia cell populations on day 11 and 14, quantitated with imaging flow cytometry analysis. (E) Morphology of normal and β-thalassemia maturing erythroid cells at different days observed by light microscopy after Giemsa staining. (F) Imaging flow cytometry analysis of ROS levels in normal and β-thalassemia erythroblast populations on day 14 detected by staining cells with CellROX green (left panel), and percentage of CellROX-positive cells (right panel). (G) Percentage of Annexin V positive erythroblasts in normal and β-thalassemia samples on the final day of culture (day 18). The data are shown as means ± SEM. *<i>P</i> < 0.05, **<i>P</i> < 0.01 by Student’s t test.</p

A T-to-G Transversion at Nucleotide −567 Upstream of HBG2 in a GATA-1 Binding Motif Is Associated with Elevated Hemoglobin F▿

Increased fetal hemoglobin (Hb F; α2γ2) production in adults can ameliorate the clinical severity of sickle cell disease and β-thalassemia major. Thus, understanding the regulation of γ-globin gene expression and its silencing in adults has potential therapeutic implications. We studied a father and son in an Iranian-American family who had elevated Hb F levels and found a novel T-to-G transversion at nucleotide (nt) −567 of the HBG2 promoter. This mutation alters a GATA-1 binding motif to a GAGA sequence located within a previously identified silencing element. DNA-protein binding assays showed that the GATA motif of interest is capable of binding GATA-1 transcription factor in vitro and in vivo. Truncation analyses of the HBG2 promoter linked to a luciferase reporter gene revealed a negative regulatory activity present between nt −675 and −526. In addition, the T-to-G mutation at the GATA motif increased the promoter activity by two- to threefold in transiently transfected erythroid cell lines. The binding motif is uniquely conserved in simian primates with a fetal pattern of γ-globin gene expression. These results suggest that the GATA motif under study has a functional role in silencing γ-globin gene expression in adults. The T-to-G mutation in this motif disrupts GATA-1 binding and the associated repressor complex, abolishing its silencing effect and resulting in the up-regulation of γ-globin gene expression in adults