9 research outputs found

    Clonal human fetal ventral mesencephalic dopaminergic neuron precursors for cell therapy research

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    A major challenge for further development of drug screening procedures, cell replacement therapies and developmental studies is the identification of expandable human stem cells able to generate the cell types needed. We have previously reported the generation of an immortalized polyclonal neural stem cell (NSC) line derived from the human fetal ventral mesencephalon (hVM1). This line has been biochemically, genetically, immunocytochemically and electrophysiologically characterized to document its usefulness as a model system for the generation of A9 dopaminergic neurons (DAn). Long-term in vivo transplantation studies in parkinsonian rats showed that the grafts do not mature evenly. We reasoned that diverse clones in the hVM1 line might have different abilities to differentiate. In the present study, we have analyzed 9 hVM1 clones selected on the basis of their TH generation potential and, based on the number of v-myc copies, v-myc down-regulation after in vitro differentiation, in vivo cell cycle exit, TH+ neuron generation and expression of a neuronal mature marker (hNSE), we selected two clones for further in vivo PD cell replacement studies. The conclusion is that homogeneity and clonality of characterized NSCs allow transplantation of cells with controlled properties, which should help in the design of long-term in vivo experimentsThis work was supported by grants from the Spanish Ministry of Economy and Competitiveness (formerly Science and Innovation; PLE2009-0101, SAF2010-17167), Comunidad AutĂłnoma Madrid (S2011-BMD-2336), Instituto Salud Carlos III (RETICS TerCel, RD06/0010/0009) and European Union (Excell, NMP4-SL-2008-214706). This work was also supported by an institutional grant from Foundation RamĂłn Areces to the Center of Molecular Biology Severo Ocho

    <i>v-myc</i> integrations and downregulation upon differentiation. A)

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    <p>Southern blot showing the number of integrated copies of v-<i>myc</i> vector in each clone and cellular lines used as controls. Human fibroblasts (Fib), negative control; clonal human forebrain neural stem cells (hNS1), known to have only one insertion <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052714#pone.0052714-Villa3" target="_blank">[35]</a>; polyclonal hVM1-BclX<sub>L</sub> cell line (HB) expected to show multiple integrations since it is composed of a library of clones. <b>B</b><b><i>)</i></b> Examples of Western blots showing v-<i>myc</i> protein detection. Samples of total protein were extracted under proliferation (Div) and after 10 days of differentiation (d10) conditions for each clone and control cell lines. Note that the clonal hNS1 cells downregulate vector expression whereas the level of v-myc protein is still high in the HB cells after 10d of differentiation. <b>C)</b><i>v-myc</i> protein signal in the blots was quantified using Image J software. The degree of downregulation (10 d/Div) is represented. The extent of down-regulation of v-<i>myc</i> protein obtained for hVM1-Bcl-X<sub>L</sub> cells (HB) was 8.3% and was taken as the minimum acceptable down-regulation that a clone should have. Thus, clones with a downregulation over 8.3% were considered. The value obtained for the downregulation of the clonal forebrain hNS1 cell line, 34.7%, would be the ideal or maximal downregulation.</p

    Cessation of cell division <i>in vivo</i>. A–H)

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    <p>Epifluorescence images of HuNu and Ki-67 immunofluorescences in sections of transplanted animals, two months post-grafting. <b>A–D)</b> Low magnification images comparing the transplants of polyclonal hVM1-Bcl-X<sub>L</sub> cells (A, B) and a representative clone (# 30; C, D). HuNu allows for the identification of all human cells in the rat section and Ki-67 labels cells engaged in the cell cycle. Note the difference in graft volume between the polyclonal cells and the clone, and that the transplant of hVM1-Bcl-X<sub>L</sub> cells looks heavily stained for Ki-67. Scale bar in D, 1 mm, valid for A-D. CC, corpus callosum; LV, lateral ventricle. <b>E–H)</b> Higher magnification view of two representative clones, showing poor (clone # 28; E, F) and good (clone # 32; G, H) downregulation of Ki-67. Scale bar: 40 ”m, valid for E-H. <b>I–J)</b> Details of neurons differentiated in the transplants of clone 32, immunostained for hNSE (I) or TH (J). Scale bar in J: 20 ”m, valid for I, J.</p

    Quantitative analysis of cell cycle exit in transplanted hVM1 cells.

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    <p>The photomicrographs are z-stack projections of sections stained for HuNu and Ki-67 through transplants of control hVM1 and hVM1-Bcl-X<sub>L</sub> cells, compared to one of the selected clones (# 30). The images in the right-hand column are high magnifications of the boxed areas to better appreciate individual nuclear staining. Scale bar corresponds to 500 ”m (left three columns of images), and 20 ”m (right column, high-power views). The histogram at the bottom shows the percentage of human cells (HuNu<sup>+</sup>) engaged in cell cycle (Ki-67<sup>+</sup>). Note that Bcl-X<sub>L</sub> does not change the percentage of proliferating cells, and that both clones are different form the polyclonal lines. Data are expressed as the mean±SEM; *** p<0.001; * p<0.05; ns p>0.05, Student t-Test.</p

    Analysis of Ki-67 and Nestin expression downregulation, and appearance of neuronal markers following differentiation.

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    <p>Ki-67 (<b>A</b>) and Nestin (<b>D</b>) immunofluorescence of the control hVM1-Bcl-X<sub>L</sub> cells (HB) and representative clones under division (Div) and following differentiation (d10). In both cases, examples are shown of a clone not showing proper downregulation (#3, 28), and of clones showing good cell cycle exit and disappearance of the NSC marker Nestin (#2, 32). <b>B</b> and <b>E</b>) Quantitative raw data are shown for control HB cells and all clones. <b>C</b> and <b>F</b>) Percentage of reduction in the number of positive cells for each marker. <b>G</b>) Immunofluorescence for ÎČ-III-tubulin and TH of the control hVM1-Bcl-X<sub>L</sub> cells (HB) and representative clones after differentiation (d10). Note the difference in neurogenic and DAn generation capacity for the different clones (<b>H</b>). Clones # 32, 2 and 13 generate more TH<sup>+</sup> cells than hVM1-Bcl-X<sub>L</sub> cells. Clones # 1, 15 and 30 are close to this level. Nuclei were counterstained with Hoechst 33258 (blue). Scale bar: 20 ”m, valid for all panels.</p

    Global account of the results obtained for the different criteria used.

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    <p>Polyclonal = hVM1-Bcl-XL cells. Scoring is presented from low values (−), to one or two (+) signs, being (++) the best result for each parameter. For the in vivo reduction of Ki- 67 staining, grafts were scored for 1 to 3, being 3 the best score indicating cell cycle exit.</p

    Assessment of drug-induced rotation. A)

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    <p>Amphetamine-induced net ipsilateral rotations before (PRE) and 2 months after transplantation (POST). Note the trend for a partial recovery observed in the majority of animal groups. Since the present work required examination of many clones, the number of animals per group was kept to a minimum (3–5) and thus non-significant trends are observed. <b>B)</b> Clone 30 transplants were twice as effective than the polyclonal hVM1-Bcl-X<sub>L</sub> cel grafts in inducing rotation compensation.</p

    Mapping European Association of Urology Guideline Practice Across Europe: An Audit of Androgen Deprivation Therapy Use Before Prostate Cancer Surgery in 6598 Cases in 187 Hospitals Across 31 European Countries

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