Laboratory, Salk Institute for Biological Studies, La Jolla, California,. USA. Upon correction of the genetic defect, induced pluripotent stem cells (iPS) from ...
SELECTED ORAL PRESENTATIONS 1
INSERM, Nice, France, 2INSERTECH, Haifa, Israel, 3Karolinska Institutet, Stockholm, Sweden, 4Radboud University, Nidjeden, The Netherlands Ectodermal dysplasia is a group of congenital syndromes affecting a variety of ectodermal derivatives. Among them, ectrodactyly, ectodermal dysplasia and cleft lip/palate (EEC) syndrome is caused by single point mutations in the p63 gene, which controls epidermal development and homeostasis. Phenotypic defects of the EEC syndrome include skin defects and limbal stem cell deficiency. In this study, we designed a novel cellular model that recapitulated major embryonic defects related to EEC. Fibroblasts from healthy donors and EEC patients carrying two different point mutations in the DNA binding domain of p63 were reprogrammed into induced pluripotent stem cell (iPSC) lines. EEC-iPSC from both patients showed early ectodermal commitment into K18 + cells but failed to further differentiate into K14 + cells (epidermis/limbus) or K3/ K12 + cells (corneal epithelium). APR-246 (PRIMA-1MET), a small compound that restores functionality of mutant p53 in human tumor cells, could revert corneal epithelial lineage commitment and reinstate normal p63-related signaling pathway. This study illustrates the relevance of iPSC for p63 related disorders. This unique modelserves to characterize the abnormal molecular circuitry of this disease and paves the way for future therapy of EEC.
OR028 Generation of Disease-free iPS Cells from Fanconi Anemia Mice with a Hypomorphic Mutation in Brca2. V Moleiro1, S Navarro1, ML Lozano1, R Chinchon1, E Almarza1, O Quintana-Bustamante1, P Rio1, G Mostoslavsky6, T Maetzig2, B Schiedlmeier2, U Modlich2, M Galla2, E Samper5, JC Segovia1, A Raya7, G Gu¨enechea1, C Baum2, JC Izpisua-Belmonte3,8, JA Bueren1 1
Hematopoietic Gene Therapy Division. Centro de Investigaciones Energe´ticas, Medioambientales y Tecnolo´gicas (CIEMAT) and Centro de Investigacio´n Biome´dica en Red de Enfermedades Raras (CIBERER), Madrid, Spain, 2Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany, 3Center of Regenerative Medicine in Barcelona, Barcelona, Spain, 4Centro de Investigacio´n Biome´dica en Red de Bioingenierı´a, Biomateriales y Nanomedicina (CIBER-BBN), Barcelona, Spain, 5Department of Regenerative Cardiology, Fundacio´n Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid, Spain, 6Center for Regenerative Medicine, Boston University School of Medicine, Boston, Massachusetts, USA, 7Institucio´ Catalana de Recerca i Estudis Avanc¸ats (ICREA), Barcelona, Spain, 8Gene Expression Laboratory, Salk Institute for Biological Studies, La Jolla, California, USA Upon correction of the genetic defect, induced pluripotent stem cells (iPS) from Fanconi anemia (FA) patients and FA mouse models can be efficiently generated and differentiated, in vitro, into hematopoietic progenitors. As mutations in the BRCA2/ FANCD1 gene have specific defects in homology directed recombination, here we have investigated the generation of iPS cells from mouse fibroblasts with a hypomorphic mutation in Brca2 (Brca2D27/D27). Defects in Brca2 implied that the reprogramming process was extremely inneficient. However, when Brca2D27/D27 MEFs were gene-complemented with a BRCA2lentiviral vector (LV) and then transduced with a reprogramming
A41 excisable polycistronic lentiviral vector, several iPSC clones were generated. iPSC clones were then transduced with an integrationdeficient LV carrying the Cre-recombinase to excise the reprogramming cassette. Gene-corrected Brca2D27/D27 iPSC clones efficiently expressed the hBRCA2 transgene and did not reveal the characteristic FA phenotype. Cells from the hematopoietic lineage could be generated from these iPSC clones, even though with a lower efficacy compared to mWT or mES cells. Additionally, no engraftment in irradiated Brca2D27/D27 recipients could be demonstrated after IV infusion of Brca2D27/D27 iPSC-derived hematopoietic grafts. Genetic analyses performed by aCGH in these samples showed the presence of genetic abnormalities which might account for the limited engraftment ability of these cells. Our studies evidence the efficacy of the cell reprogramming technology to generate disease-free iPSCs, and also reveal the limitations existing nowadays to generate safe and functional iPSC-derived hematopoietic stem cells to be used in cell therapy approaches.
OR029 Mitotic correction of trisomy 21 by disomic cell segregation occurred in human pluripotent stem cells from Down Syndrom M Valentin1,2, L Tosca1,3, O Fe´raud1, N Oudrhiri1, E Gobbo1, F Griscelli1,4, D Luton2, M Mitjavila-Garcia1, G Tachdjian1,3, A Bennaceur-Griscelli1,5 1
INSERM UMR-S 935, ESTeam Paris-Sud, Villejuif, France, Hoˆpital Beaujon, APHP, Service of gynecology, Paris, France, 3 Hoˆpitaux Universitaire Paris-Sud, APHP Service of histology and embryology, Clamart, France, 4Universite´ Paris Descartes, Paris, France, 5Hoˆpitaux Universitaires Paris-Sud, APHP, Service of Hematology, Villejuif, France 2
Down Syndrome (DS) is a common aneuploı¨d genetic disorder and only 1–2% of DS harbor a germinal or somatic postmitotic mosaI¨cism with less severe phenotypic manifestations. Autologous cell-replacement therapies after correction of trisomy 21 would be a promising therapeutic strategy. We have generated DS-iPSC from three amniotic fluid cell donors harboring a complete trisomy 21 without mosaic. Trisomy 21 was confirmed by karyotype and FISH. PloI¨dy was followed in cultured iPSC by CGHa. We observed upon iterative passages ( > 30), that one DS-iPSC line has completely lost a surnumerary chr. 21 and a disomic iPSC has been derived (PB6.1). This event was reproducible in 3 independent culture experiments. Microsatellites of chr.21 in PB6, PB6.1 and parental AFC were identical which eliminate the hypothesis of a contamination with an unrelated iPSC. Presence of chr.Y in PB6.1 by FISH eliminate a maternal contaminating cells in the initial AFC sample. SNP on ch21p confirm that persistent chr.21 originate from maternal and paternal genome. There is no heterozygosity loss. CGHa (135K) in PB6.1 compared to PB6 revealed a minor new CNV with a Dup 12q24.33 277,55Kb (including POLE, PXMP2, PGAM5, ANKLE2, GOLGA3, CHFR, ZNF605 genes) and a DupYp11.32 167,11Kb (SHOX gene). This study, is the first demonstration that IPSC can reproduce in-vitro a spontaneously correction of trisomy 21 by a post-mitotic chromosome mis-segregation mechanism. Generation of related isogenic disomic iPSC should rescue the phenotype of disease and will allow to evaluate consequences of DS phenotypic reversion in by a potential autologous normal substitution cell therapy.
