Broadening the spectrum of human embryonic stem cell Lines from PGD embryos with genetic disorder: Lines with Marfan, FSHD and Fragile X syndrome

Ileana Mateizel, Mieke Geens, Lindsey Van Haute, Claudia Spits, Bing Chen, Josiane Van Der Elst, Ingeborg Liebaers, Karen Sermon

Onderzoeksoutput: Meeting abstract (Journal)


Introduction: Despite the increasing number of human embryonic stem cells (hESC) lines reported worldwide, the derivation of new lines is currently still required in order to broaden the genetic diversity of the existing cell lines.
HESC derived from preimplantation embryos after PGD and carrying a mutation for a monogenic disease can offer the opportunity of an in vitro model of the disease because animal models are not fully representative and experiments on humans are restricted. Furthermore, the affected lines may be
used as a readily accessible source for pharmacogenetic tests and/or in vitro gene therapy experiments.
Here, we report the derivation of four new hESC lines originated from PGD embryos diagnosed as carriers of mutations for monogenic disorders: Marfan syndrome, fascio-scapulohumeral dystrophy (FSHD) and Fragile X syndrome.
Materials and methods: Embryos at day 3 post ICSI were subjected to PGD by PCR to test for the mutations causing the different disorders. Embryos found to be carrying the mutations were excluded for transfer and donated for research when informed written consent was obtained. At day 6 post ICSI,
the affected embryos were subjected to immunosurgery and the isolated ICMs were plated on mitomycin-inactivated CF1 mouse fibroblasts. Culture was done further in serum-free hESC media and the passaging was performed each 5-7 days. Characterization of stem cell properties was done at the level of
morphology, cell surface markers, gene expression and teratoma formation capacity. G-banding was used for karyotyping and the presence of mutations in the hESC lines were confirmed.
Results: Four hESC lines were derived from PGD embryos: one line carrying the c.266G.T mutation in the FBN1 gene, causing Marfan syndrome (VUB08_MFS), one line carrying a deletion in the D4Z4 locus causing
FSHD syndrome (VUB09_FSHD) and two lines carrying an expansion of approximately 2000 CGGs in the FMR1 gene, involved in Fragile X Syndrome (VUB11_FXS and VUB13_FXS).
All the established lines present a compact colony structure, cells with a high nucleus-to-cytoplasm ratio and prominent nucleoli, features considered characteristic of hES cells. The cells from all four lines were successfully frozenthawed and passaged on.
Immunocytochemistry was used to analyse the expression of surface markers that characterize undifferentiated hES cells. Colonies of undifferentiated cells were shown to be positive for SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. In addition, gene expression analysis revealed that all lines expressed genes regarded as characteristic to hESC: POU5F1, SOX2, REX1, NANOG, GDF3, DNMT3B, LIN28, and NPM1. Histological analysis of the teratomas obtained after the transplantation of the hESC into SCID-mice, confirmed the capacity of the cells to differentiate into representatives of all three germ layers: endoderm, mesoderm and ectoderm. For the analysis of G-banding karyotype, 20 metaphases were read and showed a normal 46XX karyotype at early passages for all four lines.
The lines are currently at passage 38 (VUB08_MFS), 25 (VUB09_FSHD), 50 and 37 for VUB11_FXS and VUB13_FXS, respectively.
Conclusions: VUB09_FSHD is, according to our knowledge, the first hESC line reported as being affected with FSHD. HESC lines carrying the mutation for a genetic disease can serve as an important in vitro model for the genetic disorders they carry. Unraveling the mechanisms associated with human genetic disorders can lead to new therapeutic treatments and interventions in the future.
Human biodiversity necessitates the archiving of a wide panel of affected stem cell lines.
Originele taal-2English
Pagina's (van-tot)169
Aantal pagina's1
TijdschriftHuman Reproduction
StatusPublished - 2008
EvenementUnknown - Stockholm, Sweden
Duur: 21 sep 200925 sep 2009


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