Role of astroglia in Down's syndrome revealed by patient-derived human-induced pluripotent stem cells - PubMed (original) (raw)

Peng Jiang 2, Haipeng Xue 3, Suzanne E Peterson 4, Ha T Tran 4, Anna E McCann 5, Mana M Parast 6, Shenglan Li 7, David E Pleasure 8, Louise C Laurent 9, Jeanne F Loring 9, Ying Liu 3, Wenbin Deng 10

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Role of astroglia in Down's syndrome revealed by patient-derived human-induced pluripotent stem cells

Chen Chen et al. Nat Commun. 2014.

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Abstract

Down's syndrome (DS), caused by trisomy of human chromosome 21, is the most common genetic cause of intellectual disability. Here we use induced pluripotent stem cells (iPSCs) derived from DS patients to identify a role for astrocytes in DS pathogenesis. DS astroglia exhibit higher levels of reactive oxygen species and lower levels of synaptogenic molecules. Astrocyte-conditioned medium collected from DS astroglia causes toxicity to neurons, and fails to promote neuronal ion channel maturation and synapse formation. Transplantation studies show that DS astroglia do not promote neurogenesis of endogenous neural stem cells in vivo. We also observed abnormal gene expression profiles from DS astroglia. Finally, we show that the FDA-approved antibiotic drug, minocycline, partially corrects the pathological phenotypes of DS astroglia by specifically modulating the expression of S100B, GFAP, inducible nitric oxide synthase, and thrombospondins 1 and 2 in DS astroglia. Our studies shed light on the pathogenesis and possible treatment of DS by targeting astrocytes with a clinically available drug.

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Figures

Figure 1

Figure 1. Generation and neural differentiation of DS iPSCs.

(a) A schematic procedure for directed and spontaneous differentiation of DS iPSCs to neurons and astroglia. Insets (from left to right): representatives bright-field images showing the embryoid bodies (EBs), neural rosette, neurosphere and differentiated neurons, and astroglia under directed and spontaneous differentiation conditions. Scale bars, 200 μm. (b,c) Representatives of iPSCs derived from DS patients expressing pluripotent markers Oct4 and SSEA4, as well as Nanog and Tra-1-81. (d) Representative of DS iPSC-derived NPCs expressing Pax6 and Nestin. (e,f) Representatives of βIII-tubulin+ neurons and A2B5+ glial progenitors derived from DS NPCs under directed differentiation conditions. (g) Representatives of astroglia differentiated under directed astroglial differentiation condition from control (Cont) and DS iPSCs expressing CD44 and vimentin, as well as S100B and GFAP. (h) Representatives of βIII-tubulin+ neurons and S100B+ astroglia derived from DS and Cont NPCs under spontaneous differentiation conditions. Scale bars, 50 μm. (i,j) Quantification of pooled data from Cont and DS lines showing the percentage of βIII-tubulin+ neurons and S100B+ astroglia derived from DS and Cont NPCs (_n_=3–5 from each cell line), and the length of the longest neurites of neurons (_n_=10 from each cell line) under spontaneous differentiation conditions. Student’s _t_-test, *P<0.05 and **P<0.01. Blue, 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI)-stained nuclei. Data are presented as mean±s.e.m.

Figure 2

Figure 2. Identification and correction of pathological phenotypes of DS astroglia.

(a1–6) qPCR analysis of S100B, GFAP, iNOS, TSP-1, TSP-2 and NFE2L2 mRNA expression in DS and control (Cont) astroglia. (a7) Quantification of nitrite/nitrate concentration in ACM collected from DS and Cont astroglia. One-way analysis of variance (ANOVA) test, ♣P<0.05, ♣♣P<0.01 and♣♣♣P<0.001, comparison between two DS astroglia with Cont 1 astroglia. #P<0.05,##P<0.01 and###P<0.001, comparison between two DS astroglia with Cont 2 astroglia. Student’s t_-test, *P<0.05, *P<0.01 and *♣_P<0.001, comparison between two DS astroglia or two Cont astroglia. _n_=3–4 for each cell line. (b) Representative and quantification of ROS production in Cont and DS astroglia. Green fluorescence marks cells that undergo oxidation. Student’s _t_-test, **P<0.01._n_=3–4 from each cell line. (c) Quantification of pooled data showing the proliferation rate of Cont and DS astroglia. Student’s _t_-test, **P<0.01._n_=3–4 from each cell line. (d) Glutamate uptake analysis showing that both DS and Cont astroglia were capable of glutamate uptake. Notice that DS astroglia show glutamate uptake at a higher rate at the 30- and 60-min time point than Cont astroglia. Student’s _t_-test, **P<0.01 and ***P<0.001, _n_=4 from each cell line. (e) Representatives showing intracellular uptake of the BLOCK-iT Fluorescent Oligo at 24 h after transfection of DS astroglia. Scale bar, 50 μm. (f) qPCR analysis of pooled data showing the expression of S100B gene in DS astroglia at 48 h after transfection with Cont and S100B siRNA. Student’s _t_-test, **P<0.01, _n_=3–5 from each cell line. (g) Representatives and quantification of pooled data showing ROS production in DS astroglia determined at 48 h after transfection with Cont and S100B siRNA. Student’s _t_-test, *P<0.05, _n_=5 from each cell line. (h,i) Quantification of pooled data showing the concentration of nitrite/nitrate in the ACM and proliferation rate of DS astroglia at 48 h after transfection with Cont and S100B siRNA. Student’s_t_-test, *P<0.05, _n_=3–4 from each cell line. (j) qPCR analysis of S100B, iNOS and _NEF2L2_mRNA expression in DS1 astroglia after the treatment of resveratrol, cucurmin or minocycline for 72 h. One-way ANOVA test, *P<0.05, **P<0.01 and ***P<0.001; _n_=3–5 for each group. (k) Quantitative analysis of the proliferation rate of DS1 and 2 astroglia after the treatment of minocycline. Student’s _t_-test, *P<0.05, _n_=3–4 for each cell line. (l) qPCR analysis of GFAP, TSP-1 and TSP-2 mRNA expression in DS astroglia after the treatment of minocycline. Student’s _t_-test, *P<0.05 and **P<0.01; _n_=3–4 for each group. Data are presented as mean±s.e.m.

Figure 3

Figure 3. The effects of DS astroglia on the DS NPC differentiation and DS neuron survival.

(a) Representatives of DS NPC differentiated into βIII-tubulin+ neurons and S100B+ astroglia under spontaneous differentiation condition in the presence of DS ACM, control (Cont) ACM, DS S100BsiRNA ACM and DS-Mino ACM. (b) βIII-tubulin and activated caspase3 co-staining of DS neurons cultured with different ACM. Blue, 4′,6-diamidino-2-phenylindole dihydrochloride(DAPI)-stained nuclei. Scale bars, 50 μm. (c,d) Quantification of pooled data showing the percentage of βIII-tubulin+ neurons and S100B+ astroglia derived from DS NPCs (_n_=3–4 from each cell line), and the length of the longest neurites of neurons (_n_=10 from each cell line) under spontaneous differentiation conditions in the presence of different ACM. (e) Quantification of pooled data showing the percentage of βIII-tubulin+ and activated caspase3+ cells among the groups with different treatments (_n_=3–5 from each cell line). One-way analysis of variance test, *P<0.05 and **P<0.01. Data are presented as mean±s.e.m. NS, not significant.

Figure 4

Figure 4. Electrophysiological properties of DS neurons.

(a–c) Quantification of membrane capacitance, input resistance (_R_in) and resting membrane potential (RMP) recorded from control (Cont) neurons (Neu), DS neurons, Cont neurons fed with Cont ACM or DS ACM, and DS neurons fed with DS ACM, Cont ACM or DS-Mino ACM. One-way analysis of variance test, *P<0.05. _n_=10. (d) Summarized data showing that more neurons display synaptic activity when fed with Cont ACM or DS-Mino ACM than those without being fed with ACM or fed with DS ACM. (e) Representative tracing showing that synaptic activities were recorded from Cont neurons fed with Cont ACM, DS neurons fed with Cont ACM or DS-Mino ACM, but not from Cont neuron alone or Cont neurons fed with DS ACM, and DS neuron alone or DS neuron fed with DS ACM. (f) Representative tracing showing at RMP, DS neurons fed with Cont ACM or DS-Mino ACM fire action potentials more robustly than DS neurons alone.

Figure 5

Figure 5. The effects of DS astroglia on the maturation of voltage-gated ion channels.

(a) Representative tracings showing that the inward sodium currents (_I_Na) were recorded from a DS neuron (left panel). The_I_Na can be blocked by 1 μM TTX (middle panel). The tracing of right panel was obtained by digitally subtracting the middle panel from the left panel to show the _I_Na component. The_I_Na was elicited by a series of depolarizing voltage steps (inset, from −70 to −50 mV) after a prepulse to −100 mV for 100 ms. Similar results were observed from 10 other cells. (b) Representative tracings showing potassium currents (_I_K) recorded from a DS neuron in the presence of TTX. Left panel, the overall _I_Krecorded with voltage clamp at voltages from −70 to −50 mV (inset, 200 ms duration, 20 mV increments) preceded by a prepulse conditioning potential of −80 mV, 300 ms. With a prepulse to −80 mV, the overall _I_K includes_I_KA and sustained outward current IKD. Middle panel, _I_KD recorded with voltage clamp at voltages from −70 to −50 mV (inset, 200 ms duration, 20 mV increments) preceded by a prepulse conditioning potential of +20 mV, 300 ms. The prepulse to +20 mV inactivates _I_KA component. Right panel, the _I_KA component obtained by digitally subtracting the I_KD, middle panel from the_I_K, left panel. (c–e) The_I_–_V relationship of _I_Na,_I_KD and _I_KA recorded from DS neurons and control (Cont) neurons. Pooled data, _n_=10 for each group. (f) Representative tracings showing the _I_Na,_I_KD and _I_KA recorded from Cont neurons cultured with DS ACM and Cont ACM, and DS neurons cultured with DS ACM, Cont ACM and DS-Mino ACM. (g–i) _I_–_V_relationship of _I_Na, _I_KD and_I_KA recorded from DS and Cont neurons fed with different ACM. The current densities of _I_Na,_I_KD and _I_KA recorded from neurons fed with DS ACM were smaller than those recorded from neurons fed with Cont ACM and DS-Mino ACM; one-way analysis of variance test, *P<0.05, comparison between Cont Neu+DS ACM group with other groups fed with Cont ACM and DS-Mino ACM.#P<0.05 comparison between DS Neu+DS ACM group with other groups fed with Cont ACM and DS-Mino ACM. _n_=10 for each group. Data are presented as mean±s.e.m.

Figure 6

Figure 6. Gene expression analysis of DS and control astroglia.

(a) Dendrogram showing that two control astroglia (Cont 1 and 2) and two DS astroglia (DS 1 and 2) cluster closer to each other, respectively. (b–d) Heatmaps focusing on gene transcripts encoding antioxidants and key factors in the reactive oxidative stress (b), and factors secreted by astroglia that have roles in synapse formation (c), and neurogenesis and maturation of neurons (d). High expressions relative to mean are coloured red. Low expressions are coloured green.

Figure 7

Figure 7. Pathological phenotypes observed in astroglia generated from di- and trisomic DS iPSC lines.

(a) Representative images showing that Di-DS3 astroglia and Tri-DS3 astroglia express CD44, vimentin, S100B and GFAP, and also exhibit disomy and trisomy of HAS21 by fluorescence in situ_hybridization (FISH) analysis, respectively. (b) qPCR analysis of_S100B, GFAP, TSP-1, iNOS and_TSP-2_ mRNA expression in control (Cont) 1 and 2 astroglia, Di-DS3 astroglia and Tri-DS3 astroglia with or without the treatment of minocycline. Student’s_t_-test, *P<0.05, **P<0.01, and ***P<0.001, comparison between Tri-DS3 Astro with other individual group, _n_=3–4 for each group. (c) Representative images showing NPCs from Di-DS3 and Tri-DS3 iPSCs differentiated into βIII-tubulin+ neurons and S100B+ astroglia under spontaneous differentiation condition. (d) Representative images showing DS NPCs differentiated into βIII-tubulin+ neurons and S100B+ astroglia in the presence or absence of Di-DS3 ACM or Tri-DS3 ACM. (e) Representative images of βIII-tubulin+ and activated caspase3+ cells among the groups with treatment of Di-DS3 ACM, Tri-DS3 ACM and Tri-DS3-Mino ACM. Scale bars, 50 μm (in all the images). Blue, 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI)-stained nuclei. (f) Quantification of βIII-tubulin+ neurons and S100B+ astroglia derived from Di-DS3 and Tri-DS3 NPCs (_n_=3–4 from each cell line), and the length of the longest neurites of neurons (_n_=10 from each cell line) under spontaneous differentiation condition. (g) Quantification of βIII-tubulin+ neurons and S100B+ astroglia derived from DS NPCs (_n_=3–4 from each cell line), and the length of the longest neurites of neurons (_n_=10 from each cell line) in the presence of Di-DS3 ACM or Tri-DS3 ACM. (h) Quantification of pooled data showing the percentage of βIII-tubulin+ and activated caspase3+ cells among the groups with different treatments (_n_=3–6 from each cell line). One-way analysis of variance test, *P<0.05 and **P<0.01. NS, no significant difference. (i,j) Quantification and representative tracing showing that synaptic activities were recorded from Di-DS3 neurons fed with Di-DS3 ACM, Tri-DS3 neurons fed with Di-DS3 ACM or Tri-DS3-Mino ACM, but not from Di-DS3 neurons and Tri-DS3 neurons fed with Tri-DS3 ACM.

Figure 8

Figure 8. Transplantation of astroglia into the developing brains of Rag1−/− mice.

(a) Representative images showing that at 6 weeks after transplantation, the transplanted Di-DS3 Astros and Tri-DS3 Astros were identified by human nuclei (hN) staining. Notably, the majority of the transplanted astroglia (circled by dotted lines) were found integrated into the tissue and located at the bottom of the LVs. LV, lateral ventricle; DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride. Scale bars, 500 μm. (b) Quantitative results from brain sections showing that no difference in engraftment success (hN+ cells) was noted between Di-DS3 Astro and Tri-DS3 Astro transplantation groups (_n_=4–5). NS, no significance. (c) A representative image showing that the transplanted astroglia were labelled by human CD44. Scale bars, 50 μm. (d) A representative image showing that about 50% of the transplanted hN+ cells were positive for GFAP staining. The squared area ind was enlarged in e, showing the co-localization of hN and GFAP. Scale bars, 50 and 25 μm in the original and enlarged images, respectively. (f) Some transplanted astroglia showed long GFAP+ processes, as indicated by arrowheads. The arrow indicates the cell body. Scale bar, 50 μm. (g) Representative of DCX and Ki67/nestin staining in dorsal SVZ performed on sections from control (Cont) group received PBS vehicle, groups received Di-DS3 astroglia and Tri-DS3 astroglia transplant, and a group received Tri-DS3 astroglia transplant plus minocycline treatment. Scale bars, 50 μm. (h,i) Quantitative analysis of the number of DCX+ and Ki67+ cells at dorsal SVZ in the different groups. (j) Quantitative analysis of fluorescence intensity of the nestin staining. One-way analysis of variance test,_n_=4–6; *P<0.05, **P<0.01; Data are presented as mean±s.e.m.

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