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Modelling and rescuing neurodevelopmental defect of Down syndrome using induced pluripotent stem cells from monozygotic twins discordant for trisomy 21.

Hibaoui Y, Grad I, Letourneau A, Sailani MR, Dahoun S, Santoni FA, Gimelli S, Guipponi M, Pelte MF, Béna F, Antonarakis SE, Feki A - EMBO Mol Med (2013)

Bottom Line: Here, we report the generation and characterization of induced pluripotent stem cells (iPSCs) derived from monozygotic twins discordant for trisomy 21 in order to eliminate the effects of the variability of genomic background.These defects were associated with changes in the architecture and density of neurons, astroglial and oligodendroglial cells together with misexpression of genes involved in neurogenesis, lineage specification and differentiation.Importantly, we found that targeting DYRK1A pharmacologically or by shRNA results in a considerable correction of these defects.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell Research Laboratory Department of Obstetrics and Gynecology, Geneva University Hospitals, Geneva, Switzerland.

ABSTRACT
Down syndrome (trisomy 21) is the most common viable chromosomal disorder with intellectual impairment and several other developmental abnormalities. Here, we report the generation and characterization of induced pluripotent stem cells (iPSCs) derived from monozygotic twins discordant for trisomy 21 in order to eliminate the effects of the variability of genomic background. The alterations observed by genetic analysis at the iPSC level and at first approximation in early development illustrate the developmental disease transcriptional signature of Down syndrome. Moreover, we observed an abnormal neural differentiation of Down syndrome iPSCs in vivo when formed teratoma in NOD-SCID mice, and in vitro when differentiated into neuroprogenitors and neurons. These defects were associated with changes in the architecture and density of neurons, astroglial and oligodendroglial cells together with misexpression of genes involved in neurogenesis, lineage specification and differentiation. Furthermore, we provide novel evidence that dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A) on chromosome 21 likely contributes to these defects. Importantly, we found that targeting DYRK1A pharmacologically or by shRNA results in a considerable correction of these defects.

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Schematic representation of Twin-N and Twin-DS parental fibroblasts reprogramming into Twin-N-iPSCs and Twin-DS-iPSCs using OCT4, SOX2, KLF4 and c-MYC genesPhase contrast images of Twin-N-iPSCs and Twin-DS-iPSCs growing on feeder cells.Immunofluorescence staining of Twin-N-iPSC and Twin-DS-iPSC lines for pluripotency markers NANOG, OCT4, SSEA4, TRA1-60 and TRA1-80.qRT-PCR of pluripotency-related genes; NANOG, OCT4, SOX2, LIN28 and ZFP42 ( REX1). Data are represented as mean ± s.e.m. from n = 3.DNA methylation profile of OCT4 and NANOG promoters. The global percentage of methylated cytosines (% Me) is indicated (open and closed circles indicate unmethylated and methylated CpGs, respectively).
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fig01: Schematic representation of Twin-N and Twin-DS parental fibroblasts reprogramming into Twin-N-iPSCs and Twin-DS-iPSCs using OCT4, SOX2, KLF4 and c-MYC genesPhase contrast images of Twin-N-iPSCs and Twin-DS-iPSCs growing on feeder cells.Immunofluorescence staining of Twin-N-iPSC and Twin-DS-iPSC lines for pluripotency markers NANOG, OCT4, SSEA4, TRA1-60 and TRA1-80.qRT-PCR of pluripotency-related genes; NANOG, OCT4, SOX2, LIN28 and ZFP42 ( REX1). Data are represented as mean ± s.e.m. from n = 3.DNA methylation profile of OCT4 and NANOG promoters. The global percentage of methylated cytosines (% Me) is indicated (open and closed circles indicate unmethylated and methylated CpGs, respectively).

Mentions: Normal (Twin-N) and Down syndrome (Twin-DS) fetal fibroblasts were isolated from monozygotic twins discordant for trisomy 21 (Dahoun et al, 2008) and used to establish Twin-N-iPSCs and Twin-DS-iPSCs using OCT4, SOX2, KLF4 and c-MYC genes as previously described (Takahashi et al, 2007; Grad et al, 2011; Fig 1A and B). All iPSC lines expressed markers of pluripotent cells including NANOG, OCT4, TRA-1-60, TRA-1-81 and SSEA4 as demonstrated by immunofluorescence staining (Fig 1C) and showed alkaline phosphatase activity (supplementary Fig S1A). Quantitative (qRT-PCR) and non quantitative RT-PCR analysis demonstrated expression of selected endogenous pluripotent transcription factors including OCT4, SOX2, NANOG, LIN28 and ZFP42 ( REX1) in the generated iPSCs in contrast with the parental fibroblasts (Fig 1D and supplementary Fig S1B). As expected for cells that have acquired a pluripotent state, transgene silencing was verified after initial expansion of around 10 passages of Twin-N-iPSCs and Twin-DS-iPSCs (supplementary Fig S1B). Moreover, as in hESC-H1, OCT4 and NANOG promoter regions of Twin-N-iPSCs and Twin-DS-iPSCs were found hypomethylated in comparison with their parental fibroblasts (Fig 1E).


Modelling and rescuing neurodevelopmental defect of Down syndrome using induced pluripotent stem cells from monozygotic twins discordant for trisomy 21.

Hibaoui Y, Grad I, Letourneau A, Sailani MR, Dahoun S, Santoni FA, Gimelli S, Guipponi M, Pelte MF, Béna F, Antonarakis SE, Feki A - EMBO Mol Med (2013)

Schematic representation of Twin-N and Twin-DS parental fibroblasts reprogramming into Twin-N-iPSCs and Twin-DS-iPSCs using OCT4, SOX2, KLF4 and c-MYC genesPhase contrast images of Twin-N-iPSCs and Twin-DS-iPSCs growing on feeder cells.Immunofluorescence staining of Twin-N-iPSC and Twin-DS-iPSC lines for pluripotency markers NANOG, OCT4, SSEA4, TRA1-60 and TRA1-80.qRT-PCR of pluripotency-related genes; NANOG, OCT4, SOX2, LIN28 and ZFP42 ( REX1). Data are represented as mean ± s.e.m. from n = 3.DNA methylation profile of OCT4 and NANOG promoters. The global percentage of methylated cytosines (% Me) is indicated (open and closed circles indicate unmethylated and methylated CpGs, respectively).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3927959&req=5

fig01: Schematic representation of Twin-N and Twin-DS parental fibroblasts reprogramming into Twin-N-iPSCs and Twin-DS-iPSCs using OCT4, SOX2, KLF4 and c-MYC genesPhase contrast images of Twin-N-iPSCs and Twin-DS-iPSCs growing on feeder cells.Immunofluorescence staining of Twin-N-iPSC and Twin-DS-iPSC lines for pluripotency markers NANOG, OCT4, SSEA4, TRA1-60 and TRA1-80.qRT-PCR of pluripotency-related genes; NANOG, OCT4, SOX2, LIN28 and ZFP42 ( REX1). Data are represented as mean ± s.e.m. from n = 3.DNA methylation profile of OCT4 and NANOG promoters. The global percentage of methylated cytosines (% Me) is indicated (open and closed circles indicate unmethylated and methylated CpGs, respectively).
Mentions: Normal (Twin-N) and Down syndrome (Twin-DS) fetal fibroblasts were isolated from monozygotic twins discordant for trisomy 21 (Dahoun et al, 2008) and used to establish Twin-N-iPSCs and Twin-DS-iPSCs using OCT4, SOX2, KLF4 and c-MYC genes as previously described (Takahashi et al, 2007; Grad et al, 2011; Fig 1A and B). All iPSC lines expressed markers of pluripotent cells including NANOG, OCT4, TRA-1-60, TRA-1-81 and SSEA4 as demonstrated by immunofluorescence staining (Fig 1C) and showed alkaline phosphatase activity (supplementary Fig S1A). Quantitative (qRT-PCR) and non quantitative RT-PCR analysis demonstrated expression of selected endogenous pluripotent transcription factors including OCT4, SOX2, NANOG, LIN28 and ZFP42 ( REX1) in the generated iPSCs in contrast with the parental fibroblasts (Fig 1D and supplementary Fig S1B). As expected for cells that have acquired a pluripotent state, transgene silencing was verified after initial expansion of around 10 passages of Twin-N-iPSCs and Twin-DS-iPSCs (supplementary Fig S1B). Moreover, as in hESC-H1, OCT4 and NANOG promoter regions of Twin-N-iPSCs and Twin-DS-iPSCs were found hypomethylated in comparison with their parental fibroblasts (Fig 1E).

Bottom Line: Here, we report the generation and characterization of induced pluripotent stem cells (iPSCs) derived from monozygotic twins discordant for trisomy 21 in order to eliminate the effects of the variability of genomic background.These defects were associated with changes in the architecture and density of neurons, astroglial and oligodendroglial cells together with misexpression of genes involved in neurogenesis, lineage specification and differentiation.Importantly, we found that targeting DYRK1A pharmacologically or by shRNA results in a considerable correction of these defects.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell Research Laboratory Department of Obstetrics and Gynecology, Geneva University Hospitals, Geneva, Switzerland.

ABSTRACT
Down syndrome (trisomy 21) is the most common viable chromosomal disorder with intellectual impairment and several other developmental abnormalities. Here, we report the generation and characterization of induced pluripotent stem cells (iPSCs) derived from monozygotic twins discordant for trisomy 21 in order to eliminate the effects of the variability of genomic background. The alterations observed by genetic analysis at the iPSC level and at first approximation in early development illustrate the developmental disease transcriptional signature of Down syndrome. Moreover, we observed an abnormal neural differentiation of Down syndrome iPSCs in vivo when formed teratoma in NOD-SCID mice, and in vitro when differentiated into neuroprogenitors and neurons. These defects were associated with changes in the architecture and density of neurons, astroglial and oligodendroglial cells together with misexpression of genes involved in neurogenesis, lineage specification and differentiation. Furthermore, we provide novel evidence that dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A) on chromosome 21 likely contributes to these defects. Importantly, we found that targeting DYRK1A pharmacologically or by shRNA results in a considerable correction of these defects.

Show MeSH
Related in: MedlinePlus