Differentiation of human embryonic stem cells into hepatocytes in 2D and 3D culture systems in vitro

Human embryonic stem cells (hESCs) have enormous potential as a source of cells for cell replacement therapies and as a model for early human development. In this study we examined the differentiating potential of hESCs into hepatocytes in two- and three-dimensional (2D and 3D) culture systems. Embryoid bodies (EBs) were inserted into a collagen scaffold 3D culture system or cultured on collagen-coated dishes and stimulated with exogenous growth factors to induce hepatic histogenesis. Immunofluorescence analysis revealed the expression of albumin (ALB) and cytokeratin-18 (CK-18). The differentiated cells in 2D and 3D culture system displayed several characteristics of hepatocytes, including expression of transthyretin, alpha-1-antitrypsin, cytokeratin 8, 18, 19, tryptophan-2,3-dioxygenase, tyrosine aminotransferase, glucose-6-phosphatase (G6P), cytochrome P450 subunits 7a1 and secretion of alpha-fetoprotein (AFP) and ALB and production of urea. In 3D culture, ALB and G6P were detected earlier and higher levels of urea and AFP were produced, when compared with 2D culture. Electron microscopy of differentiated hESCs showed hepatocyte-like ultrastructure, including glycogon granules, well-developed Golgi apparatuses, rough and smooth endoplasmic reticuli and intercellular canaliculi. The differentiation of hESCs into hepatocyte-like cells within 3D collagen scaffolds containing exogenous growth factors, gives rise to cells displaying morphological features, gene expression patterns and metabolic activities characteristic of hepatocytes and may provide a source of differentiated cells for treatment of liver diseases.     

Signaling Crosstalk of FHIT,p53, and p38 in etoposide-induced apoptosis in MCF-7 cells

Fragile histidine trail (FHIT) is a tumor suppressor in response to DNA damage which has been deleted in various tumors. However, the signaling mechanisms and interactions of FHIT with regard to apoptotic proteins including p53 and p38 in the DNA damage-induced apoptosis are not well described. In the present study, we used etoposide-induced DNA damage in MCF-7 as a model to address these crosstalks. The time course study showed that the expression of FHIT, p53, and p38MAPK started after 1 hour following etoposide treatment. FHIT overexpression led to increase p53 expression, p38 activation, and augmented apoptosis following etoposide-induced DNA damage compared to wild-type cells. However, FHIT knockdown blocked p53 expression, delayed p38 activation, and completely inhibited etoposide-induced apoptosis. Inhibition of p38 activity prevented induction of p53, FHIT, and apoptosis in this model. Thus, activation of p38 upon etoposide treatment leads to increase in FHIT and p53 expression. In p53 knockdown MCF-7, the FHIT induction was hampered but p38 activation was induced in lower doses of etoposide. In p53 knockdown cells, inhibition of p38 induced FHIT expression and apoptosis. Our data demonstrated that the exposure of MCF-7 cells to etoposide increases apoptosis through a mechanism involving the activation of the p38-FHIT-p53 pathway. Moreover, our findings suggest signaling interaction for these pathways may represent a promising therapy for breast cancer.     

Neovascular age-related macular degeneration risk based on CFH, LOC387715/HTRA1, and smoking

Background

Age-related macular degeneration (AMD) is the major cause of blindness in the elderly. Those with the neovascular end-stage of disease have irreversible loss of central vision. AMD is a complex disorder in which genetic and environmental factors play a role. Polymorphisms in the complement factor H (CFH) gene, LOC387715, and the HTRA1 promoter are strongly associated with AMD. Smoking also contributes to the etiology. We aimed to provide a model of disease risk based on these factors.

Methods and Findings

We genotyped polymorphisms in CFH and LOC387715/HTRA1 in a case–control study of 401 patients with neovascular AMD and 266 controls without signs of disease, and used the data to produce genetic risk scores for the European-descent population based on haplotypes at these loci and smoking history. CFH and LOC387715/HTRA1 haplotypes and smoking status exerted large effects on AMD susceptibility, enabling risk scores to be generated with appropriate weighting of these three factors. Five common haplotypes of CFH conferred a range of odds ratios (ORs) per copy from 1 to 4.17. Most of the effect of LOC387715/HTRA1 was mediated through one detrimental haplotype (carriage of one copy: OR 2.83; 95% confidence interval [CI] 1.91–4.20), with homozygotes being at particularly high risk (OR 32.83; 95% CI 12.53–86.07). Patients with neovascular macular degeneration had considerably higher scores than those without disease, and risk of blinding AMD rose to 15.5% in the tenth of the population with highest predicted risk.

Conclusions

An individual''s risk of developing AMD in old age can be predicted by combining haplotype data with smoking status. Until there is effective treatment for AMD, encouragement to avoid smoking in those at high genetic risk may be the best option. We estimate that total absence of smoking would have reduced the prevalence of severe AMD by 33%. Unless smoking habits change or preventative treatment becomes available, the prevalence of AMD will rise as a consequence of the increasing longevity of the population.     

Assessing cranial plasticity in humans: The impact of artificial deformation on masticatory and basicranial structures

Artificial cranial deformations (ACD) are a widespread cultural practice found in numerous historical and prehistoric contexts. Their study can yield valuable insight into craniofacial growth, specifically into the interactions between neurocranial and basicranial modules. This study seeks to reinvestigate the presumed effect of ACD on basicranial and masticatory elements by applying a 3D geometric morphometric approach to CT scans. A total of 51 French and Bolivian skulls, representing anteroposterior and circumferential deformations and including undeformed individuals, were scanned, and 3D landmarks were submitted to between-group principal components analysis and two-block partial least-squares analysis. Our results illustrate changes in basicranial shape and in cranial base angles induced by ACD, as well as in masticatory geometry, namely in the relative position of the mandibular fossae. Furthermore, our findings highlight differential effects of the various deformation types, which suggest that patterns of covariation between modified vaults and their associated basicrania are more complex than previously assumed, thereby stressing the degree of plasticity in human craniofacial growth.     

The survey on cellular and tissue-engineered therapies in Europe and neighboring Eurasian countries in 2014 and 2015

Background aims

With the support of five established scientific organizations, this report, the seventh of its kind, describes activity in Europe for the years 2014 and 2015 in the area of cellular and tissue-engineered therapies, excluding hematopoietic stem cell (HSC) treatments for the reconstitution of hematopoiesis.

Anti-apoptotic effect of insulin-like growth factor (IGF)-I and its receptor in porcine preimplantation embryos derived from in vitro fertilization and somatic cell nuclear transfer

Insulin-like growth factor (IGF)-I is a receptor-mediated autocrine and/or paracrine growth and/or survival factor for mammalian embryo development. It is known to promote the growth and development of mouse preimplantation embryos. The present study was designed to investigate the effects of IGF-I (50 ng/ml), anti-IGF-I receptor antibody (50 ng/ml) and their combination on porcine preimplantation embryo development. Furthermore, the mechanism underlying the embryotropic effects of IGF-I was evaluated by monitoring the incidence of apoptosis and expression of apoptosis-related genes. In both in vitro fertilized (IVF) and somatic cell nuclear transfer (SCNT) embryos, culturing with IGF-I increased the rate of blastocyst formation and this embryotrophic effect was neutralized by culturing with IGF-I along with anti-IGF-I receptor (IGF-IR) antibody. Culturing IVF and SCNT embryos with IGF-I significantly increased the number of total cells in blastocysts and decreased the number of apoptotic nuclei. These effects of IGF-I were also neutralized by culturing with IGF-I along with anti-IGF-IR antibody. Expression of the anti-apoptotic Bcl-2 gene was increased, while expression of the pro-apoptotic Bax was decreased in both IVF and SCNT embryos cultured with IGF-I. In both IVF and SCNT embryos, anti-IGF-IR antibody along with IGF-I neutralized the effect of IGF-I on expression of Bcl-2 and Bax genes. In conclusion, the present study demonstrated that IGF-I through its specific receptors improved the developmental competence of IVF and SCNT embryos by decreasing the incidence of apoptosis and regulating apoptosis-related genes in porcine preimplantation embryos.     

Down-regulation of nestin in mesenchymal stem cells derived from peripheral blood through blocking bone morphogenesis pathway

Different signaling pathways are implicated in proliferation and differentiation of stem cells. Bone Morphogenesis Pathway (BMP) signaling was known to display an important function in osteogenic and adipogenic differentiation of mesenchymal stem cells (MSCs). In the present study, the authors investigated whether blocking BMP signaling was associated with down regulation of Nestin expression as neural stem cell marker in peripheral blood derived mesenchymal stem cells (PB-MSCs). At first, MSCs were isolated from peripheral blood by plastic adherent ability and flow cytometry analysis. After reaching the confluence, the cells were treated with medium containing Noggin as antagonist of BMP signaling upon 8 days. Real time PCR analysis indicated that the expression of Nestin was diminished in PB-MSCs by attenuating BMP signaling. The obtained results suggested that BMP signaling might have a regulatory function on the Nestin expression in mesenchymal stem cells.     

Assessment of metals in agricultural soil of surrounding areas of Urmia Lake,northwest Iran: A preliminary ecological risk assessment and source identification

The aim of this study was to quantify the levels of toxic heavy metals and sodium in topsoil of farmlands around the Urmia Lake. A total of 96 topsoil samples were collected, pre-treated, and analyzed for metals using ICP-AES.

Median concentrations of As, Cd, Cu, Ni, Pb, Zn, and Na in sampling sites were 5, 0.26, 30, 40, 13, 84, and 251 mg/kg, respectively. The enrichment factor, geoaccumulation index, and contamination factor of the metals ranked them in the order of: Na > Pb > Cu > As > Cd > Zn > Ni, indicating minor contamination for them except Na and Pb with moderate contamination. Furthermore, the spatial analysis indicated that Cd, Cu, Pb, Ni, and Zn had similar distribution patterns in the north and northwest lands of the lake. Principal component analysis revealed anthropogenic sources for Cd, Cu, Ni, Pb, and Zn in the soil, whereas, sources of As and Na were most likely related to the dust emitting from the dried bed of Urmia Lake and from a cement industry. Potential ecological risk index in 7% of the sampling sites was at serious or considerable pollution level, and Cd and Pb were identified as the main pollutants.     

Influence of HOCl…O3 and HOCl…HOCl interactions on the stability of O3(HOCl)2 complexes: a theoretical study

We have analysed the role of HOCl…O₃ and HOCl…HOCl interactions on the stability of four estimated O₃(HOCl)₂ complexes by means of ab initio molecular orbital calculations. It is predicted that the O₃(HOCl) + HOCl reaction is more energetically favourable than (HOCl)₂ + O₃ one. In all complexes, HOCl…HOCl interaction is stronger than HOCl…O₃ one. The results show that the HOCl…O₃ interaction strengthens the HOCl…HOCl one. On the other hand, O…H interaction in HOCl…O₃ moiety is strengthened when it interacts with HOCl. Quantum theory of atoms in molecules predicts that the weak interactions in O₃(HOCl)₂ complexes have electrostatic characteristic. In all complexes, the charge transfer from O₃ to (HOCl)₂ is expected from natural bond orbital analysis.     

Bioprocess engineering of <Emphasis Type="Italic">Echium italicum</Emphasis> L.: induction of shikonin and alkannin derivatives by two-liquid-phase suspension cultures

An in vitro cell suspension culture of Echium italicum was established and assayed for the production of shikonin and alkannin derivatives. Callus tissues were induced from cotyledon explants of the plant incubated onto the solidified B5 medium. A two-liquid-phase system suspension culture was then established to elicit pigments of shikonin and alkannin derivatives using liquid paraffin. The presence of liquid paraffin efficiently induced production of pigments in cultured cells. The production and/or accumulation of these compounds in the E. italicum cells was examined using fluorescence microscopy as the naphthoquinone molecules display autofluorescent properties. Phytochemical analysis of the n-hexane extract of the medium was also carried out using preparative HPLC. The chemical structure of shikonin and alkannin derivatives were characterized by UV, 1H-NMR, and 13C-NMR techniques. Based on our findings, this bioprocess engineering approach resulted in induction of shikonin and alkannin derivatives, whereupon it may be recruited for production of these important secondary metabolites.