Derivation of human embryonic stem cell lines from embryos obtained after IVF and after PGD for monogenic disorders

Human Reproduction Vol.21, No.2 pp. 503–511, 2006 doi:10.1093/humrep/dei345 Advance Access publication November 10, 2005. Derivation of human embryonic stem cell lines from embryos obtained after IVF and after PGD for monogenic disorders I.Mateizel1,*, N.De Temmerman1,*, U.Ullmann1,*, G.Cauffman1, K.Sermon1,2,5, H.Van de Velde1,3, M.De Rycke1,2, E.Degreef 4, P.Devroey1,3, I.Liebaers1,2 and A.Van Steirteghem1,3 1 Research Centre for Reproduction and Genetics, 2Centre for Medical Genetics, 3Centre for Reproductive Medicine and 4Department of Pathology, University Hospital and Medical School of the Vrije Universiteit Brussel (Dutch-speaking Brussels Free University) Laarbeeklaan 101, 1090 Brussels, Belgium 5 To whom correspondence should be addressed at: University Hospital and Medical School of the Vrije Universiteit Brussel, Research Centre for Reproduction and Genetics, Laarbeeklaan 101, 1090 Brussels, Belgium. E-mail: *These authors contributed equally to this work BACKGROUND: Human embryonic stem (hES) cells are pluripotent cells usually derived from the inner cell mass (ICM) of blastocysts. Because of their ability to differentiate into all three embryonic germ layers, hES cells represent an important material for studying developmental biology and cell replacement therapy. hES cell lines derived from blastocysts diagnosed as carrying a genetic disorder after PGD represent in vitro disease models. METHODS: ICMs isolated by immunosurgery from human blastocysts donated for research after IVF cycles and after PGD were plated in serum-free medium (except VUB01) on mouse feeder layers. RESULTS: Five hES cell lines were isolated, two from IVF embryos and three from PGD embryos. All lines behave similarly in culture and present a normal karyotype. The lines express all the markers considered characteristic of undifferentiated hES cells and were proven to be pluripotent both in vitro and in vivo (ongoing for VUB05_HD). CONCLUSIONS: We report here on the derivation of two hES cell lines presumed to be genetically normal (VUB01 and VUB02) and three hES cell lines carrying mutations for myotonic dystrophy type 1 (VUB03_DM1), cystic fibrosis (VUB04_CF) and Huntington disease (VUB05_HD). Key words: cystic fibrosis/disease models/human embryonic stem cells/Huntington disease/myotonic dystrophy Introduction Human embryonic stem (hES) cells are usually derived from the inner cell mass (ICM) of preimplantation embryos (Thomson et al., 1998). Once removed from the blastocyst, the ICM can be cultured into ES cells that have the ability to remain undifferentiated and proliferate indefinitely in vitro while maintaining the potential to differentiate into all three embryonic germ layers (Thomson et al., 1998; Reubinoff et al., 2000) and trophoblast (Gerami-Naini et al., 2004). Because of these characteristics, they are of significant interest as a renewable source of cell populations for different applications. An important field of research on hES cells focuses on the study of the mechanisms of cell differentiation and developmental biology. Another major research area on hES cells concentrates on their potentially promising use in cell replacement therapies for the treatment of degenerative human diseases. Finally, because animal models are not fully representative and experiments on humans are restricted, derivation of hES cell lines known to be carriers of a monogenic disease can offer the opportunity of an in vitro model of the disease. Those affected hES cell lines would be a readily accessible source for pharmacogenetic tests or in vitro gene therapy experiments. PGD is a procedure that allows for the detection of a genetic defect at the level of an embryo fertilized in vitro and prior to transfer in utero (Sermon et al., 2004). PGD is performed by fluorescent in situ hybridization for the diagnosis of aneuploidies or X-linked pathologies and by PCR for the diagnosis of single gene disorders. PGD embryos diagnosed as affected by monogenic diseases such as myotonic dystrophy type 1 (DM1), cystic fibrosis (CF) and Huntington disease (HD) have been used in our centre for derivation of new hES cell lines. DM1 (OMIM 160900) is an autosomal dominant disorder caused by a mutation in the dystrophia myotonica protein kinase gene (DMPK) with a prevalence of 9.3 cases per 100,000 (Siciliano et al., 2001). The symptoms are characterized by myotonia, muscular dystrophy, cataract, cardiac conductance disturbance and cognitive impairment. The molecular basis of the disorder is an unstable trinucleotide CTG repeat in the 3′untranslated region of the DMPK gene. The severity of the disease and the age of onset vary with the number of repeats: DM1 is commonly an adult onset disease, but a very high © The Author 2005. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: 503 I.Mateizel et al. number of repeats leads to the congenital form, which is usually transmitted through the mother. CF (OMIM 219700) is the most common autosomal recessive disease in Caucasians, with a carrier frequency of 5%, affecting 1 out of 2000 newborns (Davis, 2001). The early childhood onset of clinical features such as chronic bronchopulmonary infections, pancreatic insufficiency and disturbed sweat gland function is caused by mutations in the coding region of the cystic fibrosis transmembrane conductance regulator gene (CFTR). The majority of male CF patients are infertile due to a congenital bilateral absence of the vas deferens (CBAVD). In patients presenting CBAVD only, CFTR mutations are found in 80% of the patients. The intron 8 splice variant 5T (5T variant) is an example of a CFTR mutation restricted to CBAVD (Lissens et al., 1996). HD (OMIM 143100) is an autosomal dominant neurodegenerative disease associated with an increase in the length of a triplet CAG expansion present in the huntingtin gene. HD has a prevalence in the Caucasian population of 5 per 100,000 (Evers-Kiebooms et al., 2002). The classic clinical features, typically of adult onset, are progressive motor impairment, cognitive decline, chorea and seizures. The course of the disease is fatal usually 15–20 years after onset. In our centre, since July 2003, IVF embryos without any known genetic defect or PGD embryos identified as affected were donated for research and used for the derivation of hES cell lines. Here, we describe the establishment of five new hES cell lines: two from IVF embryos (VUB01 and VUB02), one from a PGD embryo identified as affected by DM1 (VUB03_DM1), one from a PGD embryo diagnosed as compound heterozygous for the F508del mutation and the 5T variant (VUB04_CF) and finally one from a PGD embryo identified as affected by HD (VUB05_HD). Materials and methods Source of embryos Human embryos at blastocyst stage that had been donated for research after IVF and PGD cycles were used for this study with the infor (...truncated)