Generation of Customized Embryonic Stem Cells. Frank Yates, Human Embryonic Stem Cell Core Facility, Paul Brousse Hospital, Villejuif, France
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1 Generation of Customized Embryonic Stem Cells Frank Yates, Human Embryonic Stem Cell Core Facility, Paul Brousse Hospital, Villejuif, France
2 Obtaining Pluripotent Stem Cells to model of genetic disorders Diseased somatic cell 1) Nuclear transfer / reprogramming 2) Genetic modifications Reprogramming (Cell fusion, factors...) Diseased zygote/embryo 3) Derivation after Preimplantation Genetic Disorders Disease Model ES cell
3 Obtaining Pluripotent Stem Cells to model of genetic disorders Normal ES cell 1) Nuclear transfer / reprogramming 2) Genetic modifications Genetic Modification (Virus, homologous recombination...) 3) Derivation after Preimplantation Genetic Disorders Disease Model ES cell
4 Genetic modification through recombinant DNA Recombinant DNA (Vectors : Virus, lipid-mediated transfection...)
5 Genetic modification through recombinant DNA
6 Heterogenous population of genetically modified hes cells
7 SIngle cell cloning of genetically homogeneous populations of genetically modified hes cells
8 Clonogenicity of Human Embryonic Stem Cell Lines 14% 14% Clonogenicity (%) 12% 10% 8% 6% 4% 2% 0% 0.57% BGO1.c (n=3) 12% 10% 8% 6% 4% 2% 0% 0.24% H9.c (n=6) Human ES cells have a very low cloning efficiency
9 Clonogenicity of Human Embryonic Stem Cell Lines Collagenase passaging (48 passages) Trypsin passaging (6 passages)
10 Clonogenicity of Human Embryonic Stem Cell Lines 14% 14% Clonogenicity (%) 12% 10% 8% 6% 4% 2% 0% 0.57% BGO1.c (n=3) 7.63% BGO1.ta (n=3) 12% 10% 8% 6% 4% 2% 0% 0.24% H9.c (n=6) 3.19% H9.ta (n=5) 35 pass. 7 pass. 48 pass. 6 pass.
11 Clonogenicity of Human Embryonic Stem Cell Lines Collagenase passaging (48 passages) Trypsin passaging (6 passages) Collagenase passaging (7 passages)
12 Clonogenicity of Human Embryonic Stem Cell Lines 14% 14% Clonogenicity (%) 12% 10% 8% 6% 4% 2% 0% 0.57% BGO1.c (n=3) 7.63% BGO1.ta (n=3) 1.44% BgO1.rc (n=4) 12% 10% 8% 6% 4% 2% 0% 3.19% 0.24% 0.15% H9.c (n=6) H9.ta (n=5) H9.rc (n=3) 35 pass. (normal karyotype) 7 pass. (abnormal karyotype) 15 pass. (abnormal karyotype) 48 pass. (normal karyotype) 6 pass. (Normal karyotype) 14 pass. (Normal karyotype)
13 Clonogenicity of Human Embryonic Stem Cell Lines - Possible to transiently increase (10x) the clonogenicity of HESC by switching culture conditions -The mechanism is independant of the karyotype of the cells It is possible to subclone hesc in a high throughput setting and select a clone of interest
14 Obtaining Pluripotent Stem Cells to model of genetic disorders 2 cells for PGD analysis 1) Nuclear transfer / reprogramming 2) Genetic modifications 8 cell stage embryo Healthy Embryo 3) Derivation after Preimplantation Genetic Disorders Diseased embryo ES cell model
15 Current list of human Embryonic Stem Cell lines obtained after Preimplantation Genetic Diagnosis Chicago, USA (Kuliev et al.) Adrenoleukodystrophia Duchenne muscular dystrophy Dystrophie musculaire Becker Steinert muscular dystrophy Fanconi A anemia Fragile X syndrome Huntington Marfan syndrome Neurofibromatosis type I Thalassemie Bruxelles, Belgium (Mateizel et al.) Steinert myotonic dystrophy Cystic fibrosis Huntington Haifa, Israel : Steinert myotonic dystrophy Van Waardenburg syndrome London, UK (Pickering et al.) Cystic fibrosis
16 Derivation of hescs as disease models Step 1: Blastocyst culture Step 2: Derivation
17 Characterization of the new line CLO1 Current work : - Expression of Oct-4, Nanog, Rex1, htert - Teratoma - In vitro differentiation potential
18 Characterization of the new line CLO1
19 Characterization of the new line CLO1 Karyotypic analysis (Probe 1pter) Trisomy 1 pter (Probe 21qter) Monosomy 21 qter
20 Perspectives Effect of monosomy 21 pter / trisomy 1 qter on the differentiation capacity Derivation of new lines with other genetic disorders
21 Daley Lab, Children s Hospital, Boston, MA George Daley Elayne Chan Thorsten Schlaeger Leah Boyer Human ES cell core facility Paul Brousse Hospital (Villejuif, France) Annelise Bennaceur-Griscelli Laure Coulombel Ali Thuran Olivier Feraud Aurelie Magniez Cytogenetics Dept (Clamart, France) G. Tachdjian N. Frydman R. Frydman C. Bas Technion Institute (Haifa, Israel) J. Itskovitz-Eldor M. Amit
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