GENERATION OF MOUSE INDUCED PLURIPOTENT STEM CELLS BY PROTEIN TRANSDUCTION.

Somatic cell reprogramming has generated enormous interest after the first report by Yamanaka and his coworkers in 2006 on the generation of induced pluripotent stem cells (iPSCs) from mouse fibroblasts. Here we report the generation of stable iPSCs from mouse fibroblasts by recombinant protein transduction (Klf4, Oct4, Sox2 and c-Myc), a procedure designed to circumvent the risks caused by integration of exogenous sequences in the target cell genome associated with gene delivery systems. The recombinant proteins were fused in frame to the GST tag for affinity purification and to the TAT-NLS polypeptide to facilitate membrane penetration and nuclear localization. We performed the reprogramming procedure on embryonic fibroblasts from inbred (C57BL6) and outbred (ICR) mouse strains. The cells were treated with purified proteins four times, at 48-hour intervals, and cultured on mitomycin C treated MEF (mouse embryonic fibroblast) cells in complete embryonic stem cell medium until colonies formed. The iPSCs generated from the outbred fibroblasts exhibited similar morphology and growth properties to embryonic stem (ESC) cells and were sustained in an undifferentiated state for more than 20 passages. The cells were checked for pluripotency-related markers (Oct4, Sox2, Klf4, cMyc, Nanog) by immunocytochemistry and by RT-PCR. The protein iPSCs (piPSCs) formed EBs and subsequently differentiated towards all three germ layer lineages. Importantly the piPSCs could incorporate into the blastocyst and led to variable degrees of chimerism in newborn mice. These data show that recombinant purified cell-penetrating proteins are capable of reprogramming mouse embryonic fibroblasts to iPSCs. We also demonstrated that the cells of the generated cell line satisfied all the requirements of bona fide mouse ESC cells: form round colonies with defined boundaries; have a tendency to attach together with high nuclear/cytoplasmic ratio; express key pluripotency markers; and are capable of in vitro differentiation into ecto-, endo-, and mesoderm, and in vivo chimera formation

Expression profiles of the pluripotency marker gene POU5F1 and validation of reference genes in rabbit oocytes and preimplantation stage embryos

<p>Abstract</p> <p>Background</p> <p>The surge in the number of gene expression studies and tendencies to increase the quality of analysis have necessitated the identification of stable reference genes. Although rabbits are classical experimental model animals, stable reference genes have not been identified for normalization. The aims of this study were to compare the expression profiles of the widely used reference genes in rabbit oocytes and preimplantation stage embryos, and to select and validate stable ones to use as reference.</p> <p>Results</p> <p>Quantitative real time PCR method was used to evaluate 13 commonly used references (<it>Actb, Gapdh, Hprt1, H2afz, Ubc, Ppia, Eef1e1, Polr2a, Tbp, G6pdx, B2m, Pgk1</it>, and <it>Ywhaz</it>) and <it>POU5F1 (Oct4) </it>genes. Expressions of these genes were examined in multiple individual embryos of seven different preimplantation developmental stages and embryo types (<it>in vivo </it>and <it>in vitro</it>). Initial analysis identified three genes (<it>Ubc, Tbp</it>, and <it>B2m) </it>close to the detection limit with irregular expression between the different stages. As variability impedes the selection of stable genes, these were excluded from further analysis. The expression levels of the remaining ten genes, varied according to developmental stage and embryo types. These genes were ranked using the geNorm software and finally the three most stable references (<it>H2afz, Hprt1</it>, and <it>Ywhaz</it>) were selected. Normalization factor was calculated (from the geometric averages of the three selected genes) and used to normalize the expressions of <it>POU5F1 </it>gene. The results showed the expected expression patterns of the POU5F1 during development.</p> <p>Conclusion</p> <p>Compared to the earlier studies with similar objectives, the comparison of large number of genes, the use of multiple individual embryos as compared to pools, and simultaneous analyses of <it>in vitro </it>and <it>in vivo </it>derived embryo samples were unique approaches in our study. Based on quantification, pattern and geNorm analyses, we found the three genes (<it>H2afz, Hprt1</it>, and <it>Ywhaz</it>) to be the most stable across developmental stages and embryo types, and the geometric averages of these genes can be used for appropriate normalization.</p

Promoter analysis of the rabbit POU5F1 gene and its expression in preimplantation stage embryos

Background: The POU5F1 gene encodes the octamer-binding transcription factor-4 (Oct4). It is crucial in the regulation of pluripotency during embryonic development and widely used as molecular marker of embryonic stem cells (ESCs). The objective of this study was to identify and to analyse the promoter region of rabbit POU5F1 gene; furthermore to examine its expression pattern in preimplantation stage rabbit embryos. [br/] Results: The upstream region of rabbit POU5F1 was subcloned sequenced and four highly conserved promoter regions (CR1-4) were identified. The highest degree of similarity on sequence level was found among the conserved domains between rabbit and human. Among the enhancers the proximal enhancer region (PE-1A) exhibited the highest degree of homology (96.4%). Furthermore, the CR4 regulator domain containing the distal enhancer (DE-2A) was responsible for stem cell-specific expression. Also, BAC library screen revealed the existence of a processed pseudogene of rabbit POU5F1. The results of quantitative real-time PCR experiments showed that POU5F1 mRNA was abundantly present in oocytes and zygotes, but it was gradually reduced until the activation of the embryonic genome, thereafter a continuous increase in POU5F1 mRNA level was observed until blastocyst stage. By using the XYClone laser system the inner cell mass (ICM) and trophoblast portions of embryos were microdissected and examined separately and POU5F1 mRNA was detected in both cell types. [br/] Conclusion: In this study we provide a comparative sequence analysis of the regulatory region of rabbit POU5F1 gene. Our data suggest that the POU5F1 gene is strictly regulated during early mammalian development. We proposed that the well conserved CR4 region containing the DE-2A enhancer is responsible for the highly conserved ESC specific gene expression. Notably, we are the first to report that the rabbit POU5F1 is not restricted to ICM cells only, but it is expressed in trophoblast cells as well. This information may be well applicable to investigate further the possible phylogenetic role and the regulation of POU5F1 gene

Average gene expression stability values of the candidate reference genes in the derived and produced embryo samples as calculated by geNorm software and ranking made based on the relative stability values

<p><b>Copyright information:</b></p><p>Taken from "Expression profiles of the pluripotency marker gene and validation of reference genes in rabbit oocytes and preimplantation stage embryos"</p><p>http://www.biomedcentral.com/1471-2199/9/67</p><p>BMC Molecular Biology 2008;9():67-67.</p><p>Published online 28 Jul 2008</p><p>PMCID:PMC2507718.</p><p></p

Seamless Connectivity System for Intelligent Public Transportation Systems: Architecture and Mechanism Design

Providing ubiquitous connectivity to the passengers of public transportation vehicles is an important goal of the communication system designers in the context of fast development of the intelligent transportation systems and of the Future Internet communication technologies. This paper proposes the architecture of a connectivity system for public transportation communication services, the architecture design being considered on three distinct levels: system, functional and platform level. The proposed system architecture specifies a minimal set of entities required to implement the envisaged connectivity solution and based on a functional analysis the subsystems and modules are derived. By mapping the functional architecture on the hardware components intended to be used the platform architecture is developed. The paper proposes also the design of the mechanisms which implement the inter-process communications, perform the acquisition and handling of the context information and implement a distributed information system characterizing the heterogeneous networking environment. For other mechanisms, like decision and mobility management, the design principles are described. In order to validate the proposed architecture design and to check the correct functioning of the various subsystems and modules a few experimental tests are presented

Amelioration of Hyperbilirubinemia in Gunn Rats after Transplantation of Human Induced Pluripotent Stem Cell-Derived Hepatocytes

SummaryHepatocyte transplantation has the potential to cure inherited liver diseases, but its application is impeded by a scarcity of donor livers. Therefore, we explored whether transplantation of hepatocyte-like cells (iHeps) differentiated from human induced pluripotent stem cells (iPSCs) could ameliorate inherited liver diseases. iPSCs reprogrammed from human skin fibroblasts were differentiated to iHeps, which were transplanted into livers of uridinediphosphoglucuronate glucuronosyltransferase-1 (UGT1A1)-deficient Gunn rats, a model of Crigler-Najjar syndrome 1 (CN1), where elevated unconjugated bilirubin causes brain injury and death. To promote iHep proliferation, 30% of the recipient liver was X-irradiated before transplantation, and hepatocyte growth factor was expressed. After transplantation, UGT1A1+ iHep clusters constituted 2.5%–7.5% of the preconditioned liver lobe. A decline of serum bilirubin by 30%–60% and biliary excretion of bilirubin glucuronides indicated that transplanted iHeps expressed UGT1A1 activity, a postnatal function of hepatocytes. Therefore, iHeps warrant further exploration as a renewable source of hepatocytes for treating inherited liver diseases

Peripheral CD4+ cell prevalence and pleuropulmonary manifestations in systemic lupus erythematosus patients.

INTRODUCTION: Systemic lupus erythematosus (SLE) is an autoimmune disease involving several organs, including the lungs. Previous results confirmed changes of peripheral T cell subsets in lupus patients; however no data are available about their possible relationship with pulmonary involvement. OBJECTIVE: To determine pulmonary manifestations and potential relationship in changes of peripheral CD4+ T cell subsets. METHODS: Patients with SLE (N = 28) were enrolled in complex pulmonary examination. Patients were divided into groups with pleuropulmonary manifestations (SLEpulmN = 13 age: 44.9 +/- 3.3 years, female: male = 11:2) or without (SLEcN = 15 age: 27.2 +/- 3.7 years, female: male = 12:3). Peripheral blood was taken for T helper (Th)1, Th2, Th17, CD4+CD25hi+ and regulatory T (Treg: CD4+CD25hi+ CD127-) cell analysis from SLE patients and healthy volunteers (controls, N = 40). RESULTS: SLEpulm patients were older, had more pulmonary symptoms and significantly decreased pO2 as compared to SLEc group. Ventilatory disorder was present in 92% of SLEpulm patients, with significantly decreased lung volumes, signs of airway involvement and decrease in DLco. Significant increase in Th1/Th2, while decrease in Th17/Treg ratios was present in all SLE compared to controls. In SLEpulm CD4+CD25hi+ subset without changes in Treg number was significantly increased as compared to SLEc and this subgroup of T cell showed significant positive correlation with dynamic lung function parameters and DLco (p < 0.05). CONCLUSION: In lupus patients pleuropulmonary manifestations are prevalent and lung function and blood gas measurements should be regularly performed in the daily clinical assessment. Significant increase of activated CD4+CD25hi+ T cells, but not Treg is associated with decreased lung function parameters in SLEpulm patients