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Cited by 122Down syndrome and intellectual disability researchPluripotent Stem Cells ResearchNeurogenesis and neuroplasticity mechanisms

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Modeling Down Syndrome with Patient iPSCs Reveals Cellular and Migration Deficits of GABAergic Neurons

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Huo, Hai-Qin, Qu, Zhuang-Yin, Yuan, Fang, Ma, Lixiang, Yao, Lin, Xu, Min, Hu, Yao, Ji, Jing, Bhattacharyya, Anita, Zhang, Su-Chun, Liu, Yan·2018·Stem Cell Reports
DOI 10.1016/j.stemcr.2018.02.001PMC PMC5998838
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Abstract

The brain of Down syndrome (DS) patients exhibits fewer interneurons in the cerebral cortex, but its underlying mechanism remains unknown. By morphometric analysis of cortical interneurons generated from DS and euploid induced pluripotent stem cells (iPSCs), we found that DS GABA neurons are smaller and with fewer neuronal processes. The proportion of calretinin over calbindin GABA neurons is reduced, and the neuronal migration capacity is decreased. Such phenotypes were replicated following transplantation of the DS GABAergic progenitors into the mouse medial septum. Gene expression profiling revealed altered cell migratory pathways, and correction of the PAK1 pathway mitigated the cell migration deficit in vitro. These results suggest that impaired migration of DS GABAergic neurons may contribute to the reduced number of interneurons in the cerebral cortex and hippocampus in DS patients.

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Figure 1 Trisomic iPSC-Derived GABAergic Interneurons Show Abnormal Gene Expression In Vitro (A) Experimental outline. Euploid and trisomy iPSC- or hESC-derived MGE progenitors differentiated to GABAergic interneurons or transplanted to the SCID mouse brain. (B) GABAergic interneurons were generated from euploid and trisomy iPSC lines at day 35. Scale bar, 50 μm. (C) Proportion of GABA+, TH+, and ChAT+ neurons among total (TUJ1+) neurons derived from each cell line at day 35. At least 800 cells were counted from random selected field in each experiment, n ≥ 3 for independent experiments; bar graph presents the mean ± SEM. (D) Representative images of euploid and trisomy GABAergic interneurons generated from each cell lines in vitro. Scale bar, 100 μm. (E) Quantification of soma size, branches, and longest neurite length of euploid and DS GABAergic interneurons at days 3, 7 and 10 after plating. At least 500 cells were counted from random selected field in each experiment, n ≥ 3 for independent experiments; bar graphs present the mean ± SD. (F) High-content measurement workflow. After differentiation from DS iPSCs, GABAergic interneurons were plated on 96-well plates at a density of 2,000 cells per well. (G) Quantification of soma size, branches, and neurite length of euploid and DS GABAergic interneurons at day 7 after plating. At least 2,500 cells were analyzed for each cell line, n ≥ 3 for independent experiments; bar graphs present the mean ± SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.
Figure 1 Trisomic iPSC-Derived GABAergic Interneurons Show Abnormal Gene Expression In Vitro (A) Experimental outline. Euploid and trisomy iPSC- or hESC-derived MGE progenitors differentiated to GABAergic interneurons or transplanted to the SCID mouse brain. (B) GABAergic interneurons were generated from euploid and trisomy iPSC lines at day 35. Scale bar, 50 μm. (C) Proportion of GABA+, TH+, and ChAT+ neurons among total (TUJ1+) neurons derived from each cell line at day 35. At least 800 cells were counted from random selected field in each experiment, n ≥ 3 for independent experiments; bar graph presents the mean ± SEM. (D) Representative images of euploid and trisomy GABAergic interneurons generated from each cell lines in vitro. Scale bar, 100 μm. (E) Quantification of soma size, branches, and longest neurite length of euploid and DS GABAergic interneurons at days 3, 7 and 10 after plating. At least 500 cells were counted from random selected field in each experiment, n ≥ 3 for independent experiments; bar graphs present the mean ± SD. (F) High-content measurement workflow. After differentiation from DS iPSCs, GABAergic interneurons were plated on 96-well plates at a density of 2,000 cells per well. (G) Quantification of soma size, branches, and neurite length of euploid and DS GABAergic interneurons at day 7 after plating. At least 2,500 cells were analyzed for each cell line, n ≥ 3 for independent experiments; bar graphs present the mean ± SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.CC BY-NC-ND · PubMed Central
Figure 2 Cellular and Migratory Analysis of GABAergic Interneurons In Vitro (A) The expression of CR in euploid and trisomy iPSC-derived neurons at day 40. Scale bar, 100 μm. (B) Quantification of CR at days 20, 40, and 80 in euploid and trisomy cell lines in vitro. At least 2,000 cells were counted from random selected field in each experiment, n ≥ 3 for independent experiments; bar graph presents the mean ± SEM. (C) The euploid and trisomy iPSC-derived neurons expressed CB at day 40. Scale bar, 100 μm. (D) Quantification of CB immunostaining analyses at days 20 and 40 in euploid and trisomy cell lines in vitro. At least 3,000 cells were counted from random selected field in each experiment, n ≥ 3 for independent experiments; bar graph presents the mean ± SEM. (E) Representative phase image of euploid and trisomy GABAergic interneurons that migrated from neurospheres. Scale bar, 100 μm. (F) Quantification of numbers and migration length of euploid and trisomy GABAergic neurons. Euploid groups include DS2U, IMR90-4, and H9. At least 12 neurospheres were plated, and 200 cells were counted in each experiment, n ≥ 3 for independent experiments; bar graphs present the mean ± SD. (G) Representative images of scratch assay in euploid and trisomy cell lines. Scale bar, 100 μm. (H) Quantification of the nuclei presented on the scratched area after 3 days for euploid and trisomy cell lines. n ≥ 3 for independent experiments; graph presents the mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. NS, not significant.
Figure 2 Cellular and Migratory Analysis of GABAergic Interneurons In Vitro (A) The expression of CR in euploid and trisomy iPSC-derived neurons at day 40. Scale bar, 100 μm. (B) Quantification of CR at days 20, 40, and 80 in euploid and trisomy cell lines in vitro. At least 2,000 cells were counted from random selected field in each experiment, n ≥ 3 for independent experiments; bar graph presents the mean ± SEM. (C) The euploid and trisomy iPSC-derived neurons expressed CB at day 40. Scale bar, 100 μm. (D) Quantification of CB immunostaining analyses at days 20 and 40 in euploid and trisomy cell lines in vitro. At least 3,000 cells were counted from random selected field in each experiment, n ≥ 3 for independent experiments; bar graph presents the mean ± SEM. (E) Representative phase image of euploid and trisomy GABAergic interneurons that migrated from neurospheres. Scale bar, 100 μm. (F) Quantification of numbers and migration length of euploid and trisomy GABAergic neurons. Euploid groups include DS2U, IMR90-4, and H9. At least 12 neurospheres were plated, and 200 cells were counted in each experiment, n ≥ 3 for independent experiments; bar graphs present the mean ± SD. (G) Representative images of scratch assay in euploid and trisomy cell lines. Scale bar, 100 μm. (H) Quantification of the nuclei presented on the scratched area after 3 days for euploid and trisomy cell lines. n ≥ 3 for independent experiments; graph presents the mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. NS, not significant.CC BY-NC-ND · PubMed Central
Figure 3 Survival and Differentiation of DS GABAergic Interneurons in the Mouse Brain (A) GABAergic interneuron progenitors were injected into medial septum of SCID mice. The white dashed lines represent endogenous neuronal projections to the hippocampus. Scale bar, 500 μm. (B) Grafted human (HN+) cells from both euploid and trisomy neurons survived in the medial septum 6 months after transplantation. Scale bar, 100 μm. (C) Quantification of human cell numbers in the graft in euploid and trisomy groups show no significant difference for the survival grafted cells (9,522–15,055 and 5,758–20,617 HN+ cells were counted, n = 4; bar graph presents the mean ± SEM). (D) Representative images of euploid and trisomy grafted human GABAergic interneurons in the mouse brain. The red lines illustrate the primary branches, and the blue lines illustrate the secondary branches. Scale bar, 20 μm. (E) Quantification of soma size and its distribution, neurite arborization, and longest neurites of grafted euploid and trisomy GABAergic interneurons (n = 4; bar graphs present the mean ± SEM). (F) Five representative neuronal types for the grafted human neurons. (G) Distribution of the five types of grafted GABAergic neurons (n = 4; bar graph presents the mean ± SEM). (H and I) Representative images of grafted human GABAergic interneuron subtypes, including calbindin (CB), calretinin (CR), somatostatin (SST), and parvalbumin (PV). The human PV+ neurons are observed out of the graft. Scale bars, 50 μm. (J) Proportion of GABAergic interneuron subtypes, including CB, SST, CR, and PV, for euploid and trisomy groups (n = 4; bar graph presents the mean ± SEM). (K) Quantification of CB+ neurons soma size in euploid and trisomy grafts. There was no significant difference between two groups (n = 3; bar graph presents the mean ± SEM). (L and M) DS SST+ neuron (L) and CR+ neurons (M) show smaller soma size than euploid control. Euploid group refers to DS2U and Trisomy group refers to DS1 (n = 4; bar graphs present the mean ± SEM). ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.
Figure 3 Survival and Differentiation of DS GABAergic Interneurons in the Mouse Brain (A) GABAergic interneuron progenitors were injected into medial septum of SCID mice. The white dashed lines represent endogenous neuronal projections to the hippocampus. Scale bar, 500 μm. (B) Grafted human (HN+) cells from both euploid and trisomy neurons survived in the medial septum 6 months after transplantation. Scale bar, 100 μm. (C) Quantification of human cell numbers in the graft in euploid and trisomy groups show no significant difference for the survival grafted cells (9,522–15,055 and 5,758–20,617 HN+ cells were counted, n = 4; bar graph presents the mean ± SEM). (D) Representative images of euploid and trisomy grafted human GABAergic interneurons in the mouse brain. The red lines illustrate the primary branches, and the blue lines illustrate the secondary branches. Scale bar, 20 μm. (E) Quantification of soma size and its distribution, neurite arborization, and longest neurites of grafted euploid and trisomy GABAergic interneurons (n = 4; bar graphs present the mean ± SEM). (F) Five representative neuronal types for the grafted human neurons. (G) Distribution of the five types of grafted GABAergic neurons (n = 4; bar graph presents the mean ± SEM). (H and I) Representative images of grafted human GABAergic interneuron subtypes, including calbindin (CB), calretinin (CR), somatostatin (SST), and parvalbumin (PV). The human PV+ neurons are observed out of the graft. Scale bars, 50 μm. (J) Proportion of GABAergic interneuron subtypes, including CB, SST, CR, and PV, for euploid and trisomy groups (n = 4; bar graph presents the mean ± SEM). (K) Quantification of CB+ neurons soma size in euploid and trisomy grafts. There was no significant difference between two groups (n = 3; bar graph presents the mean ± SEM). (L and M) DS SST+ neuron (L) and CR+ neurons (M) show smaller soma size than euploid control. Euploid group refers to DS2U and Trisomy group refers to DS1 (n = 4; bar graphs present the mean ± SEM). ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.CC BY-NC-ND · PubMed Central
Figure 4 Migration of Human GABAergic Interneurons (A) Sagittal sections showing migration path and migrated cells (HN+) in olfactory bulb area. Scale bars, 100 μm. (B) Quantification of migrated human cells in olfactory bulb (n = 3; bar graph presents the mean ± SEM). (C) Quantification of furthest distance of cell migration to olfactory bulb from the center of the graft (n = 3; bar graph presents the mean ± SEM). (D) Sagittal sections showing presence of Stem121+ human neurites (red outline) and cells (dotted outline) from medial septum to posterior hippocampus in euploid (DS2U and H9) and trisomy groups (DS2 and 2DS3). Scale bar, 100 μm. (E) Quantification of migrated human cells in the hippocampus (n = 4; bar graph presents the mean ± SEM). (F) Quantification of furthest distance of cell migration to hippocampus from the center of the graft (n = 4; bar graph presents the mean ± SEM). ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.
Figure 4 Migration of Human GABAergic Interneurons (A) Sagittal sections showing migration path and migrated cells (HN+) in olfactory bulb area. Scale bars, 100 μm. (B) Quantification of migrated human cells in olfactory bulb (n = 3; bar graph presents the mean ± SEM). (C) Quantification of furthest distance of cell migration to olfactory bulb from the center of the graft (n = 3; bar graph presents the mean ± SEM). (D) Sagittal sections showing presence of Stem121+ human neurites (red outline) and cells (dotted outline) from medial septum to posterior hippocampus in euploid (DS2U and H9) and trisomy groups (DS2 and 2DS3). Scale bar, 100 μm. (E) Quantification of migrated human cells in the hippocampus (n = 4; bar graph presents the mean ± SEM). (F) Quantification of furthest distance of cell migration to hippocampus from the center of the graft (n = 4; bar graph presents the mean ± SEM). ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.CC BY-NC-ND · PubMed Central
Figure 5 Axonal Projection of GABAergic Neurons toward Hippocampus (A) Coronal sections showing presence of Stem121+ human neurites and cells in dorsal hippocampus from anterior to posterior in euploid and trisomy groups. Scale bar, 100 μm. (B) Schematic images illustrating how the neurites in euploid and trisomy groups that cross line 1 (the line tangent to anterior CA3) and line 2 (the line tangent to posterior tip of dentate gyrus) are counted. (C) Quantification of human neurite projection length in hippocampus in euploid and trisomy groups (n = 4; bar graph presents the mean ± SEM). (D) Quantification of distribution areas with human neurites in hippocampus (n = 4; bar graph presents the mean ± SEM). (E) Quantification of neurite numbers that cross line 1 and line 2 in euploid and trisomy groups (n = 4; bar graph presents the mean ± SEM). (F) Colabeling of grafted Stem121+ neurites/cells with GABA (green) in hippocampus. Insets are shown at high magnification. Euploid group refers DS2U and Trisomy group refers DS1. The arrowheads represent the co-labeling of Stem121+ neurites/cells with GABA. ∗∗∗p < 0.001. Scale bars, 100 μm.
Figure 5 Axonal Projection of GABAergic Neurons toward Hippocampus (A) Coronal sections showing presence of Stem121+ human neurites and cells in dorsal hippocampus from anterior to posterior in euploid and trisomy groups. Scale bar, 100 μm. (B) Schematic images illustrating how the neurites in euploid and trisomy groups that cross line 1 (the line tangent to anterior CA3) and line 2 (the line tangent to posterior tip of dentate gyrus) are counted. (C) Quantification of human neurite projection length in hippocampus in euploid and trisomy groups (n = 4; bar graph presents the mean ± SEM). (D) Quantification of distribution areas with human neurites in hippocampus (n = 4; bar graph presents the mean ± SEM). (E) Quantification of neurite numbers that cross line 1 and line 2 in euploid and trisomy groups (n = 4; bar graph presents the mean ± SEM). (F) Colabeling of grafted Stem121+ neurites/cells with GABA (green) in hippocampus. Insets are shown at high magnification. Euploid group refers DS2U and Trisomy group refers DS1. The arrowheads represent the co-labeling of Stem121+ neurites/cells with GABA. ∗∗∗p < 0.001. Scale bars, 100 μm.CC BY-NC-ND · PubMed Central
Figure 6 Transcriptional Analysis of Trisomic and Euploid GABAergic Interneurons by RNA Sequencing (A) Total number of genes and the top 30 fold-changed genes that were altered in trisomy GABAergic interneurons with an FDR of 5%. (B) Top 9 altered biological processes in trisomy GABAergic interneurons in comparison with euploid controls by using gene ontology. p ≤ 0.05. (C) The expression of representative genes involved in regulating cell morphology revealed by RNA-seq. (D) Heatmap of differentiation-related gene expression in euploid and trisomy GABAergic interneurons. (E) Cell motility-related pathways that were altered in trisomy GABAergic interneurons by using KEGG pathway enrichment. (F) A network plot of “Regulation of actin cytoskeleton” pathway genes and their connections.
Figure 6 Transcriptional Analysis of Trisomic and Euploid GABAergic Interneurons by RNA Sequencing (A) Total number of genes and the top 30 fold-changed genes that were altered in trisomy GABAergic interneurons with an FDR of 5%. (B) Top 9 altered biological processes in trisomy GABAergic interneurons in comparison with euploid controls by using gene ontology. p ≤ 0.05. (C) The expression of representative genes involved in regulating cell morphology revealed by RNA-seq. (D) Heatmap of differentiation-related gene expression in euploid and trisomy GABAergic interneurons. (E) Cell motility-related pathways that were altered in trisomy GABAergic interneurons by using KEGG pathway enrichment. (F) A network plot of “Regulation of actin cytoskeleton” pathway genes and their connections.CC BY-NC-ND · PubMed Central
Figure 7 Deregulation of Cofilin Rescued Migration Deficit of DS GABAergic Neurons (A and B) RNA-seq (A) and qRT-PCR analysis (B) of PAK1. (C and D) Representative western blots for PAK1 (C) and p-cofilin (D) in euploid and trisomy GABAergic interneurons (n ≥ 3 for independent experiments; bar graphs present the mean ± SEM). (E) Western blot analysis of PAK1 in trisomy GABAergic interneurons after treating with FRAX486 (FRAX = FRAX486, n ≥ 3 for independent experiments; bar graph presents the mean ± SEM). (F and G) Western blot analysis of p-PAK1 (F) and p-cofilin (G) in trisomy GABAergic interneurons after treating with FRAX486 (n ≥ 3 for independent experiments; bar graphs present the mean ± SEM). (H) Representative phase image of FRAX486-treated trisomy GABAergic interneuron that migrated from neurospheres. Scale bar, 100 μm. (I) Quantification of migration length for (H). At least 10 neurospheres were plated and 400 cells were counted in each experiment, n ≥ 3 for independent experiments; bar graph presents the mean ± SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. NS, not significant.
Figure 7 Deregulation of Cofilin Rescued Migration Deficit of DS GABAergic Neurons (A and B) RNA-seq (A) and qRT-PCR analysis (B) of PAK1. (C and D) Representative western blots for PAK1 (C) and p-cofilin (D) in euploid and trisomy GABAergic interneurons (n ≥ 3 for independent experiments; bar graphs present the mean ± SEM). (E) Western blot analysis of PAK1 in trisomy GABAergic interneurons after treating with FRAX486 (FRAX = FRAX486, n ≥ 3 for independent experiments; bar graph presents the mean ± SEM). (F and G) Western blot analysis of p-PAK1 (F) and p-cofilin (G) in trisomy GABAergic interneurons after treating with FRAX486 (n ≥ 3 for independent experiments; bar graphs present the mean ± SEM). (H) Representative phase image of FRAX486-treated trisomy GABAergic interneuron that migrated from neurospheres. Scale bar, 100 μm. (I) Quantification of migration length for (H). At least 10 neurospheres were plated and 400 cells were counted in each experiment, n ≥ 3 for independent experiments; bar graph presents the mean ± SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. NS, not significant.CC BY-NC-ND · PubMed Central

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Authors
Huo, H.-Q., Qu, Z.-Y., Yuan, F., Ma, L., Yao, L., Xu, M., Hu, Y., Ji, J., Bhattacharyya, A., Zhang, S.-C., & Liu, Y.
Affiliations
  • State Key Laboratory of Reproductive Medicine (CN)
  • Shanghai Medical College of Fudan University (CN)
  • University of Wisconsin–Madison (US)
  • Nanjing Medical University (CN)
  • Jiangsu Province Hospital (CN)
  • University of Wisconsin System (US)
Corresponding author
  • Zhang, Su-Chun
  • Liu, Yan
ORCID
Venue
Stem Cell Reports
Publisher
Elsevier BV
Published
2018-04
Volume
10
Issue
4
Pages
1251-1266
ISSN
2213-6711 (ISSN-L)
DOI
10.1016/j.stemcr.2018.02.001
PMC
PMC5998838
OpenAlex
W2789849905
Cited by
122
References
47
Citations by year
2026: 9 · 2025: 13 · 2024: 15 · 2023: 14 · 2022: 20 · 2021: 27 · 2020: 8 · 2019: 13 · 2018: 3
FWCI
12.16
Type
journal article
Language
en
Open access
Licence
Locations
  • Stem Cell Reports (publishedVersion, cc-by-nc-nd)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
  • National Institutes of Health (MH100031, MH099587, R21HD085288)
  • National Basic Research Program of China (2016YFC1306703)
  • Government of Jiangsu Province
  • National Natural Science Foundation of China (BK20160044, BK20160047, 81471301)
Topics
  • Health Sciences › Medicine › Public Health, Environmental and Occupational Health › Down syndrome and intellectual disability research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Pluripotent Stem Cells Research
  • Life Sciences › Neuroscience › Developmental Neuroscience › Neurogenesis and neuroplasticity mechanisms
Keywords
Down syndrome, GABAergic interneurons, neuronal migration, iPSC-derived neurons, calretinin, calbindin, cerebral cortex, hippocampus, cell morphometry
MeSH terms
Animals, Brain, Cell Differentiation, Cell Movement, Cell Survival, Down Syndrome, Gene Expression Regulation, Humans, Interneurons, Models, Biological, Somatostatin, Neurites, Gene Expression Profiling, Cell Shape, Actin Depolymerizing Factors, Mice, p21-Activated Kinases, Induced Pluripotent Stem Cells, Neural Stem Cells, GABAergic Neurons, Calbindin 2

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