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

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.

Figure 1

1. 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 genes

2. Phase contrast images of Twin-N-iPSCs and Twin-DS-iPSCs growing on feeder cells.

3. Immunofluorescence staining of Twin-N-iPSC and Twin-DS-iPSC lines for pluripotency markers NANOG, OCT4, SSEA4, TRA1-60 and TRA1-80.

4. qRT-PCR of pluripotency-related genes; NANOG, OCT4, SOX2, LIN28 and ZFP42 ( REX1). Data are represented as mean ± s.e.m. from n = 3.

5. 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).

Figure 2

1. Karyotypes of Twin-N-iPSCs and Twin-DS-iPSCs are 46, XX and 47, XX+21, respectively.

2. Ideogram and array-CGH profile showing the gain of one DNA copy of chromosome 21 in Twin-DS-iPSCs in comparison with Twin-N-iPSCs.

3. Comparison of the normalized gene expression levels mean of log2 RPKM between Twin-N-iPSCs and Twin-DS-iPSCs (HSA21 genes are shown in red and the rest of the genome in blue).

4. Principal component analysis (PCA) plot based on the normalized expression values in Twin-N-iPSCs and Twin-DS-iPSCs. The percentages represent the proportion of variance explained by each component.

5. Heat map of the normalized gene expression values in Twin-DS-iPSCs and Twin N-iPSCs for the 1204 differentially expressed genes. A negative z-score (in red) indicates low expression (below the mean) whereas a positive z-score (in green) shows high expression (above the mean). Diagram showing the proportion of genes differentially expressed between Twin-N-iPSCs and Twin-DS-iPSCs. See also supplementary Table S2.

Figure 3

1. Hematoxylin and eosin staining analysis of teratoma generated after intramuscular injection of Twin-N-iPSCs (upper panel) and Twin-DS-iPSCs (lower panel) into SCID mice. See also supplementary Fig S4.

2. Spontaneous in vitro differentiation of Twin-N-iPSCs (upper panel) and Twin-DS-iPSCs (lower panel) as embryoid bodies (EBs) in suspension culture for 4 days and as adherent cells for an additional 17 days. These EBs expressed α-SMA (mesoderm), AFP (endoderm) and β3-tubulin (ectoderm).

Figure 4

A Schematic representation of neural induction of the iPSCs into NPCs and neuronal differentiation into neurons. B–F qRT-PCR analysis of pluripotency, endodermal and mesodermal markers upon neural induction of Twin-N-iPSCs and Twin-DS-iPSCs into NPCs. The expression of neuronal, astroglial and oligodendroglial markers is also shown. Data are represented as mean ± s.e.m. For clarity, statistics are only shown at the NPC level (day 21). Ns non significant, * P < 0.05, ** P < 0.01 by Student's t-test from n = 3–5. G Proportion of SOX2/NESTIN double positive cells in NPCs derived from Twin-N-iPSCs and Twin-DS-iPSCs. See also supplementary Fig S5. H Cell proliferation analysis by Ki-67 staining of NPCs derived from Twin-N-iPSCs and Twin-DS-iPSCs. See also supplementary Fig S6. I Nuclear damage analysis of NPCs derived from Twin-N-iPSCs and Twin-DS-iPSCs (arrows, Hoechst 33342 staining). J Representative AMC fluorescence traces and quantification of caspase-3 activity in NPCs 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. See also supplementary Fig S7.

Figure 5

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.

Figure 6

A Expression and activity of DYRK1A in NPCs derived from Twin-N-iPSCs and Twin-DS-iPSCs. Data are represented as mean ± s.e.m. ** P < 0.01 by Student's t-test from n = 4. B Schematic representation for generation of NPCs from Twin-DS-iPSCs after incubation with EGCG 10 μM. C Activity of DYRK1A in NPCs derived from Twin-N-iPSCs and Twin-DS-iPSCs. The effect of 10 μM EGCG is also shown. Data are represented as mean ± s.e.m. ** P < 0.01, *** P < 0.001 by one-way ANOVA followed with Tukey's test from n = 5. D–F Effect of DYRK1A inhibition by EGCG on the number (in D), the proliferation (in E, Ki-67 staining) and cell death (in F, caspase-3 activity) of NPCs derived from Twin-DS-iPSCs. Data are represented as mean ± s.e.m. * P < 0.05, ** P < 0.01 by one-way ANOVA followed with Tukey's test from n = 4. G Schematic representation for generation of NPCs from Twin-DS-iPSCs after transduction with lentiviruses encoding shRNAs targeting DYRK1A. H–L Expression (in H) and activity (in I) of DYRK1A in NPCs derived from Twin-N-iPSCs, Twin-DS-iPSCs and Twin-DS-iPSCs with DYRK1A shRNA. Effect of DYRK1A inhibition by shRNA on the number (in J), the proliferation (in K, Ki-67 staining) and cell death (in L, caspase-3 activity) of NPCs derived from Twin-DS-iPSCs. Data are represented as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001 by one-way ANOVA followed with Tukey's test from n = 4.

Figure 7

1. Schematic representation for generation of neurons from Twin-DS-iPSCs after incubation with EGCG 10 μM.

2. Effect of DYRK1A inhibition by EGCG on the expression of neuronal markers upon neuronal differentiation of Twin-DS-iPSCs. Data are represented as mean ± s.e.m. * P < 0.05, ** P < 0.01 by one-way ANOVA followed with Tukey's test from n = 4.

3. Schematic representation for generation of neurons from Twin-DS-iPSCs after transduction with lentiviruses encoding shRNAs targeting DYRK1A.

4. Effect of DYRK1A inhibition through shRNA silencing on the expression of neuronal markers upon neuronal differentiation of Twin-DS-iPSCs. Data are represented as mean ± s.e.m. * P < 0.05, ** P < 0.01 by one-way ANOVA followed with Tukey's test from n = 4.

5. qRT-PCR of REST/NRSF, WNT7A, WNT7B, NOTCH1, NOTCH2, HES1 and DLL1 in NPCs derived from Twin-N-iPSCs and Twin-DS-iPSCs. The effect of DYRK1A inhibition by shRNA is also shown. Data are represented as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001 by one-way followed with Tukey's test from n = 4–7.