FAS-Based Cell Depletion Facilitates the Selective Isolation of Mouse Induced Pluripotent Stem Cells

Cellular reprogramming of somatic cells into induced pluripotent stem cells (iPSC) opens up new avenues for basic research and regenerative medicine. However, the low efficiency of the procedure remains a major limitation. To identify iPSC, many studies to date relied on the activation of pluripotency-associated transcription factors. Such strategies are either retrospective or depend on genetically modified reporter cells. We aimed at identifying naturally occurring surface proteins in a systematic approach, focusing on antibody-targeted markers to enable live-cell identification and selective isolation. We tested 170 antibodies for differential expression between mouse embryonic fibroblasts (MEF) and mouse pluripotent stem cells (PSC). Differentially expressed markers were evaluated for their ability to identify and isolate iPSC in reprogramming cultures. Epithelial cell adhesion molecule (EPCAM) and stage-specific embryonic antigen 1 (SSEA1) were upregulated early during reprogramming and enabled enrichment of OCT4 expressing cells by magnetic cell sorting. Downregulation of somatic marker FAS was equally suitable to enrich OCT4 expressing cells, which has not been described so far. Furthermore, FAS downregulation correlated with viral transgene silencing. Finally, using the marker SSEA-1 we exemplified that magnetic separation enables the establishment of bona fide iPSC and propose strategies to enrich iPSC from a variety of human source tissues

Coulomb dissociation of N 20,21

Neutron-rich light nuclei and their reactions play an important role in the creation of chemical elements. Here, data from a Coulomb dissociation experiment on N20,21 are reported. Relativistic N20,21 ions impinged on a lead target and the Coulomb dissociation cross section was determined in a kinematically complete experiment. Using the detailed balance theorem, the N19(n,γ)N20 and N20(n,γ)N21 excitation functions and thermonuclear reaction rates have been determined. The N19(n,γ)N20 rate is up to a factor of 5 higher at

Leber-Fibrose: Erstellung eines Modells zur Analyse lebertoxischer Substanzen und Analyse der ltbp1-abhängigen Aktivierung hepatischer Sternzellen

The liver is the central organ of metabolism and at the same time constitutes the biggest gland of the mammalian organism. Breakdown of liver function after chronic damage caused by viral infection or toxic substances implicates severe consequences for the organism. Research on the causes and the pathogenesis of liver diseases therefore is of major importance. This work adresses two questions in relation to liver fibrosis. The first part of this work deals with the expression of EGFP as a transgenic hepatocyte specific surface marker, by means of which hepatic precursors can be isolated from in vitro differentiated murine embryonic stem cells to generate functional cells for toxicological screenings. After validation of the isolation method (MACS), a reporter construct with an albumin promotor driven expression of the surface-/selection-marker was cloned and the expression specificity was analysed in vitro and in vivo. The functional expression of the transgene and its correct subcellular localization was confirmed using the hepatoma cell line HEPA-1-6. However, transfected ES cells showed basal, albumin independent expression of the transgene and even after in vitro differentiation into hepatocyte like cells did not express sufficient amounts of EGFP protein. Analysis of a transgenic mouse model generated by oocyte injection with the same reporter construct revealed that even under in vivo conditions only marginal amounts of EGFP were produced in the liver. Using a targeting strategy which aimed to knock in the EGFP-selection marker into the mouse albumin locus did not lead to the identification of positive ES cell clones. In the second part of this work, the influence of the latent transforming growth factor beta binding protein 1 (LTBP1) on the „activation“ of hepatic stellate cells (HSC) was analysed. Following „activation“ HSC transdifferentiate towards a myofibroblastic phenotype and constitute the most important fibrogenic cell type during the pathogenesis of liver fibrosis. Using a ltbp1-deficient mouse model in which both known protein isoforms LTBP1-L and LTBP1-S are missing, revealed a new ltbp1 splice variant of which analogous isoforms also exist in rats and humans. Microarray analysis was used to identify known and so far unknown marker genes for activated HSC and more importantly demonstrated that ltbp1-deficient HSC have a reduced tendency to transdifferentiate in vitro. This observation was independently confirmed in vivo using an experimental model for the induction of liver fibrosis (ligation of the common bile duct). After 4 weeks of bile duct obstruction, ltbp1-/- mice showed markedly reduced signs of liver fibrosis, i.e. less Collagen I deposition by HSC in the affected liver parenchyma. The study therefore provides insight into the role of LTBP1 during the pathogenesis of liver fibrosis and points out the clinical relevance of LTBP1, which needs to be confirmed by subsequent analysis

Disruption of the latent transforming growth factor-beta binding protein-1 gene causes alteration in facial structure and influences TGF-beta bioavailability

AbstractLatent transforming growth factor-β binding proteins are a family of extracellular matrix proteins comprising four isoforms (LTBP-1, -2, -3, -4) with different structures, tissue expression patterns and affinity for TGF-β. So far, respective knockout models have highlighted some essential functions for LTBP-2, LTBP-3 and LTBP-4, while the physiological significance of LTBP-1 is only superficially known. Here we report for the first time the generation and characterization of a mouse model lacking both the long and short LTBP-1 isoform. Surprisingly, respective mice are viable and fertile. However, detailed X-ray analysis of the skull revealed a modified facial profile. In addition, the gene disruption induces a reduced biological activity of TGF-β that became evident in an experimental model of hepatic fibrogenesis in which the LTBP-1 knockout animals were less prone to hepatic fibrogenesis. Furthermore, comparative cDNA microarray gene expression profiling of cultured hepatic stellate cells confirmed that respective nulls were less receptive to cellular activation and transdifferentiation into myofibroblasts. Therefore, we conclude that LTBP-1 has essential functions in the control of TGF-β activation

Oct4-GFP expression characteristics.

<p>A) Immunofluorescence of <i>Oct4</i>-GFP transgenic iPSC line LV1-7b cultured in non-differentiating conditions. Depicted are the <i>Oct4</i>-GFP marker, staining for OCT4 protein, a DAPI counterstain and phase contrast images. The overlay displays <i>Oct4</i>-GFP and OCT4 protein. B) The same iPSC line and analysis as in A cultured under differentiating conditions (2 day LIF withdrawal).</p

Biological Similarities Between Mesenchymal Stem Cells (Mscs) and Fibroblasts

Ghodsizad A, Voelkel T, Moebius JM, et al. Biological Similarities Between Mesenchymal Stem Cells (Mscs) and Fibroblasts. Journal of Cytology &amp; Histology. 2010;1(01)

Expression analysis of EPCAM and FAS protein in selected human cell types.

<p>A, B) Flow cytometry analysis of FAS and EPCAM expression on human foreskin fibroblasts (hFF), human umbilical vein endothelial cells (hUVEC) and two hiPSC lines. A) Representative flow cytometry analysis results. B,) Independent quantification of relative expression levels of FAS and EPCAM for the same four cell types. Stain indices (SI) are depicted to enable the comparison of expression levels for cell types displaying different levels of autofluorescence. SI were calculated as follows: (Median of labeled cells – Median of unlabeled cells)/(2× standard deviation of unlabeled cells). p values were calculated with a Student's t test. *p<0.05; **p<0.01; ***p<0.001. Scale bars show the SD for three separate experiments (alpha = 95%).</p

Excerpt of the tissue distribution of EPCAM and FAS mRNA in human cell types.

<p>An mRNA analysis based on the Genevestigator software tool was performed reflecting the tissue distribution of investigated mRNAs. Shown are selected tissues that are relatively easily accessible and have already been reprogrammed in previous reports. The “percentage of expression potential” represents the average expression of a gene across all samples of the particular cell type as compared to the sample with the highest expression (maximum level, 100%) for the particular gene [percentage of expression potential  =  average/maximum]. The number of samples that were included to calculate this average is indicated on the right side of the graph. Results are given as logarithmic (log2) heat map.</p

Expression kinetics of some candidate markers correlate with reprogramming stages.

<p>A) Expression frequencies (mean +/− SD) of candidate markers on reprogramming subpopulations were investigated by flow cytometry over time (n = 3: mean +/− SD; for SSEA1 n = 1) (also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102171#pone.0102171.s002" target="_blank">Figure S2</a>). Reprogramming subpopulations were defined as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102171#pone-0102171-g003" target="_blank">Fig. 3D</a>. B) Correlation of ITGAV, SSEA1, EPCAM and FAS with expression of OCT4 protein as analyzed by flow cytometry at day 12 p.t. C) Likewise, correlation of the selected candidate markers with the <i>Oct4</i>-GFP reporter system is shown at day 12 of reprogramming.</p