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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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A Quantitative expression of neuronal (β3-TUBULIN and MAP2), astroglial (GFAP) and oligodendroglial (OLIG2) markers after maturation of NPCs derived from Twin-N-iPSCs and Twin-DS-iPSCs into neurons, by immunofluorescence analysis. The proportion of β3-TUBULIN, GFAP and OLIG2 positive cells is also shown. Data are represented as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001 by Student's t-test from n > 4.B qRT-PCR analysis of neuronal ( TUBB3, MAP2 and FOXA2), astroglial and oligodendroglial ( GFAP, S100B, VIM, OLIG1 and OLIG2) markers upon neuronal differentiation of NPCs into neurons. Data are represented as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001 by Student's t-test from n = 4.C Representative images and quantitative analysis of neurites (either axons or dendrites) from the soma of β3-TUBULIN positive neurons derived from Twin-N-iPSCs and Twin-DS-iPSCs. Data are represented as mean ± s.e.m. *** P < 0.001 by Student's t-test from n = 4.D Quantitative analysis of the length of neurites of neurons derived from Twin-N-iPSCs and Twin-DS-iPSCs. Data are represented as mean ± s.e.m. *** P < 0.001 by Student's t-test from n = 4.E, F Quantitative analysis of MAP2 positive neurons derived from Twin-N-iPSCs and Twin-DS-iPSCs stained for SYNAPSIN1 (in E), PSD95 and GAD67 (in F). Data are represented as mean ± s.e.m. Ns non significant, * P < 0.05, ** P < 0.01 by Student's t-test from n = 3–4.G qRT-PCR of the synaptic markers SYN1, PSD95, SNPA25 and GAD67 in neurons derived from Twin-N-iPSCs and Twin-DS-iPSCs. Data are represented as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001 by Student's t-test from n = 3–5.
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fig05: A Quantitative expression of neuronal (β3-TUBULIN and MAP2), astroglial (GFAP) and oligodendroglial (OLIG2) markers after maturation of NPCs derived from Twin-N-iPSCs and Twin-DS-iPSCs into neurons, by immunofluorescence analysis. The proportion of β3-TUBULIN, GFAP and OLIG2 positive cells is also shown. Data are represented as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001 by Student's t-test from n > 4.B qRT-PCR analysis of neuronal ( TUBB3, MAP2 and FOXA2), astroglial and oligodendroglial ( GFAP, S100B, VIM, OLIG1 and OLIG2) markers upon neuronal differentiation of NPCs into neurons. Data are represented as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001 by Student's t-test from n = 4.C Representative images and quantitative analysis of neurites (either axons or dendrites) from the soma of β3-TUBULIN positive neurons derived from Twin-N-iPSCs and Twin-DS-iPSCs. Data are represented as mean ± s.e.m. *** P < 0.001 by Student's t-test from n = 4.D Quantitative analysis of the length of neurites of neurons derived from Twin-N-iPSCs and Twin-DS-iPSCs. Data are represented as mean ± s.e.m. *** P < 0.001 by Student's t-test from n = 4.E, F Quantitative analysis of MAP2 positive neurons derived from Twin-N-iPSCs and Twin-DS-iPSCs stained for SYNAPSIN1 (in E), PSD95 and GAD67 (in F). Data are represented as mean ± s.e.m. Ns non significant, * P < 0.05, ** P < 0.01 by Student's t-test from n = 3–4.G qRT-PCR of the synaptic markers SYN1, PSD95, SNPA25 and GAD67 in neurons derived from Twin-N-iPSCs and Twin-DS-iPSCs. Data are represented as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001 by Student's t-test from n = 3–5.

Mentions: When NPCs were further induced to mature into neurons, we found a decreased expression of β3-TUBULIN and MAP2, and an increase of GFAP, VIMENTIN, S100B, OLIG1 and OLIG2 markers in Twin-DS-iPSC-derived cells when compared with Twin-N-iPSC-derived cells, as revealed by immunofluorescence staining and qRT-PCR (Fig 5A and B). These results strongly support an impaired neurogenesis together with a shift from neuronal to astroglial and oligodendroglial phenotype in Twin-DS-iPSC-derived cells. Notably, this shift was still observed even when a greater number of NPCs was induced to mature into neurons which rules out the possibility that the reduced number of NPCs derived from Twin-DS-iPSCs could lead to the observed shift from neuronal to astroglial and oligodendroglial phenotype (unpublished observations). Quantification of neurite (either axons or dendrites) branching from soma of β3-TUBULIN positive neurons showed that the mean number of branches was approximately twofold lower in neurons derived from Twin-DS-iPSCs than those derived from Twin-N-iPSCs (Fig 5C). Thus, neurons derived from Twin-DS-iPSCs demonstrated reduced length of neurites (Fig 5D).


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)

A Quantitative expression of neuronal (β3-TUBULIN and MAP2), astroglial (GFAP) and oligodendroglial (OLIG2) markers after maturation of NPCs derived from Twin-N-iPSCs and Twin-DS-iPSCs into neurons, by immunofluorescence analysis. The proportion of β3-TUBULIN, GFAP and OLIG2 positive cells is also shown. Data are represented as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001 by Student's t-test from n > 4.B qRT-PCR analysis of neuronal ( TUBB3, MAP2 and FOXA2), astroglial and oligodendroglial ( GFAP, S100B, VIM, OLIG1 and OLIG2) markers upon neuronal differentiation of NPCs into neurons. Data are represented as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001 by Student's t-test from n = 4.C Representative images and quantitative analysis of neurites (either axons or dendrites) from the soma of β3-TUBULIN positive neurons derived from Twin-N-iPSCs and Twin-DS-iPSCs. Data are represented as mean ± s.e.m. *** P < 0.001 by Student's t-test from n = 4.D Quantitative analysis of the length of neurites of neurons derived from Twin-N-iPSCs and Twin-DS-iPSCs. Data are represented as mean ± s.e.m. *** P < 0.001 by Student's t-test from n = 4.E, F Quantitative analysis of MAP2 positive neurons derived from Twin-N-iPSCs and Twin-DS-iPSCs stained for SYNAPSIN1 (in E), PSD95 and GAD67 (in F). Data are represented as mean ± s.e.m. Ns non significant, * P < 0.05, ** P < 0.01 by Student's t-test from n = 3–4.G qRT-PCR of the synaptic markers SYN1, PSD95, SNPA25 and GAD67 in neurons derived from Twin-N-iPSCs and Twin-DS-iPSCs. Data are represented as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001 by Student's t-test from n = 3–5.
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fig05: A Quantitative expression of neuronal (β3-TUBULIN and MAP2), astroglial (GFAP) and oligodendroglial (OLIG2) markers after maturation of NPCs derived from Twin-N-iPSCs and Twin-DS-iPSCs into neurons, by immunofluorescence analysis. The proportion of β3-TUBULIN, GFAP and OLIG2 positive cells is also shown. Data are represented as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001 by Student's t-test from n > 4.B qRT-PCR analysis of neuronal ( TUBB3, MAP2 and FOXA2), astroglial and oligodendroglial ( GFAP, S100B, VIM, OLIG1 and OLIG2) markers upon neuronal differentiation of NPCs into neurons. Data are represented as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001 by Student's t-test from n = 4.C Representative images and quantitative analysis of neurites (either axons or dendrites) from the soma of β3-TUBULIN positive neurons derived from Twin-N-iPSCs and Twin-DS-iPSCs. Data are represented as mean ± s.e.m. *** P < 0.001 by Student's t-test from n = 4.D Quantitative analysis of the length of neurites of neurons derived from Twin-N-iPSCs and Twin-DS-iPSCs. Data are represented as mean ± s.e.m. *** P < 0.001 by Student's t-test from n = 4.E, F Quantitative analysis of MAP2 positive neurons derived from Twin-N-iPSCs and Twin-DS-iPSCs stained for SYNAPSIN1 (in E), PSD95 and GAD67 (in F). Data are represented as mean ± s.e.m. Ns non significant, * P < 0.05, ** P < 0.01 by Student's t-test from n = 3–4.G qRT-PCR of the synaptic markers SYN1, PSD95, SNPA25 and GAD67 in neurons derived from Twin-N-iPSCs and Twin-DS-iPSCs. Data are represented as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001 by Student's t-test from n = 3–5.
Mentions: When NPCs were further induced to mature into neurons, we found a decreased expression of β3-TUBULIN and MAP2, and an increase of GFAP, VIMENTIN, S100B, OLIG1 and OLIG2 markers in Twin-DS-iPSC-derived cells when compared with Twin-N-iPSC-derived cells, as revealed by immunofluorescence staining and qRT-PCR (Fig 5A and B). These results strongly support an impaired neurogenesis together with a shift from neuronal to astroglial and oligodendroglial phenotype in Twin-DS-iPSC-derived cells. Notably, this shift was still observed even when a greater number of NPCs was induced to mature into neurons which rules out the possibility that the reduced number of NPCs derived from Twin-DS-iPSCs could lead to the observed shift from neuronal to astroglial and oligodendroglial phenotype (unpublished observations). Quantification of neurite (either axons or dendrites) branching from soma of β3-TUBULIN positive neurons showed that the mean number of branches was approximately twofold lower in neurons derived from Twin-DS-iPSCs than those derived from Twin-N-iPSCs (Fig 5C). Thus, neurons derived from Twin-DS-iPSCs demonstrated reduced length of neurites (Fig 5D).

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