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

Chen C, Jiang P, Xue H, Peterson SE, Tran HT, McCann AE, Parast MM, Li S, Pleasure DE, Laurent LC, Loring JF, Liu Y, Deng W - Nat Commun (2014)

Bottom Line: DS astroglia exhibit higher levels of reactive oxygen species and lower levels of synaptogenic molecules.Transplantation studies show that DS astroglia do not promote neurogenesis of endogenous neural stem cells in vivo.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.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biochemistry and Molecular Medicine, School of Medicine, University of California, Davis, California 95817, USA [2] Institute for Pediatric Regenerative Medicine, Shriners Hospitals for Children, Sacramento, California 95817, USA [3] Department of Neurology, Institute of Neurology, Tianjin General Hospital, Tianjin Medical University, Tianjin 300070, China [4].

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.

No MeSH data available.


Related in: MedlinePlus

Generation and neural differentiation of DS iPSCs.(a) A schematic procedure for directed and spontaneous differentiationof 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 underdirected and spontaneous differentiation conditions. Scale bars,200 μm. (b,c) Representatives of iPSCsderived from DS patients expressing pluripotent markers Oct4 and SSEA4, as well asNanog and Tra-1-81.(d) Representative of DS iPSC-derived NPCs expressingPax6 andNestin.(e,f) Representatives of βIII-tubulin+ neurons andA2B5+ glial progenitors derived from DS NPCs under directed differentiationconditions. (g) Representatives of astroglia differentiated underdirected astroglial differentiation condition from control (Cont) and DSiPSCs expressing CD44 andvimentin, as well asS100B andGFAP. (h)Representatives of βIII-tubulin+ neurons and S100B+ astroglia derived from DSand Cont NPCs under spontaneous differentiation conditions. Scale bars,50 μm. (i,j) Quantification of pooleddata from Cont and DS lines showing the percentage of βIII-tubulin+ neurons andS100B+ astrogliaderived from DS and Cont NPCs (n=3–5 from each cell line),and the length of the longest neurites of neurons (n=10 from eachcell line) under spontaneous differentiation conditions.Student’s t-test, *P<0.05 and**P<0.01. Blue, 4′,6-diamidino-2-phenylindoledihydrochloride (DAPI)-stained nuclei. Data are presented asmean±s.e.m.
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f1: Generation and neural differentiation of DS iPSCs.(a) A schematic procedure for directed and spontaneous differentiationof 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 underdirected and spontaneous differentiation conditions. Scale bars,200 μm. (b,c) Representatives of iPSCsderived from DS patients expressing pluripotent markers Oct4 and SSEA4, as well asNanog and Tra-1-81.(d) Representative of DS iPSC-derived NPCs expressingPax6 andNestin.(e,f) Representatives of βIII-tubulin+ neurons andA2B5+ glial progenitors derived from DS NPCs under directed differentiationconditions. (g) Representatives of astroglia differentiated underdirected astroglial differentiation condition from control (Cont) and DSiPSCs expressing CD44 andvimentin, as well asS100B andGFAP. (h)Representatives of βIII-tubulin+ neurons and S100B+ astroglia derived from DSand Cont NPCs under spontaneous differentiation conditions. Scale bars,50 μm. (i,j) Quantification of pooleddata from Cont and DS lines showing the percentage of βIII-tubulin+ neurons andS100B+ astrogliaderived from DS and Cont NPCs (n=3–5 from each cell line),and the length of the longest neurites of neurons (n=10 from eachcell line) under spontaneous differentiation conditions.Student’s t-test, *P<0.05 and**P<0.01. Blue, 4′,6-diamidino-2-phenylindoledihydrochloride (DAPI)-stained nuclei. Data are presented asmean±s.e.m.

Mentions: To establish an in vitro human cellular model for DS and to investigateneuron-astrocyte interactions, we first generated DS hiPSC lines using thecanonical ‘Yamanaka’ reprogramming method by transducingDS patients’ fibroblasts (Coriell Medical Institute) withretroviruses encoding OCT4,SOX2, KLF4 and c-MYC (Supplementary Fig. 1A). The age-matched hiPSClines from healthy individuals were used as controls. We then differentiated theDS and control hiPSCs to neurons and astroglia via directed or spontaneousdifferentiation procedures shown in Fig. 1a. The hiPSClines expressed pluripotent makers OCT4, SSEA4, NANOG and TRA1-81 (Fig. 1b,c), andwere able to form teratomas that showed structures corresponding to three germlayers (Supplementary Fig. 1B). TheiPSCs and fibroblasts had distinct gene expression pattern, as demonstrated byanalyses of their gene expression profiles (Supplementary Fig. 1C,D). As shown in Supplementary Fig. 1E, thepluripotency of the iPSCs was also evidenced by the results of PluriTest, analgorithm built upon a global gene expression database of a total of 264 PSClines (223 hESC (human embryonic stem cell) and 41 iPSC lines), which has beenused to predict pluripotency accurately and effectively20. Two ofthe iPSC lines generated from DS patients DS1 and DS2 (Supplementary Table 1) maintained a stabletrisomic chromosome 21 karyotype during serial passaging and after neuraldifferentiation (Supplementary Fig.1F), and thus were first used in this study. The control and DS hiPSClines generated NPCs at high efficiency, as indicated by expressing NPC markers,Pax6 and Nestin (Fig. 1dand Supplementary Fig. 2A).Subsequently, under directed neuronal differentiation condition, neuronalprogenitors were further selected and cultured in the presence of neurotrophicfactors brain-derived neurotrophicfactor (BDNF) and glialcell-derived neurotrophic factor (GDNF) (Fig. 1a).Both control and DS hiPSC-derived NPCs were efficiently induced to generateneurons (>85%; Fig. 1e and Supplementary Fig. 2B,C). In parallel, underdirected astroglial differentiation condition by adding bone morphogenetic protein 4(BMP4; Fig.1a)21, the NPCs started to express glial precursormarker A2B5 at early stage (Fig. 1f), and later generatedastroglia after 20 days in culture, as identified by astroglial markersglial fibrillary acidicprotein (GFAP) and S100B (>95%; Fig. 1g and Supplementary Fig. 2D,E). Nearly allthe hiPSC-derived astroglia also expressed CD44, a marker used to identify astrocyte-restrictedprecursor cells, consistent with our recent study on astroglial differentiationof hESCs22, and vimentin, a major cytoskeletal protein expressed in immatureastrocytes23 (Fig. 1g). The robustco-expression of CD44/vimentin and GFAP/S100Bindicated that the majority of hiPSC-derived astroglia were immature, ratherthan mature astrocytes, which better mimic early developmental stages of the DSpathology in the human brain. No significant difference was observed in theefficiency of neuronal and astroglial differentiation between DS and controlhiPSC lines (Supplementary Fig.2B–E) under the directed differentiation conditions. Inaddition, similar to hESC-derived astroglia21, all hiPSCastroglial preparations expressed mRNAs encoding the astrocyte-specificglutamate transporters, glutamate-aspartatetransporter (GLAST) and glutamatetransporter-1 (GLT-1), as detected by quantitative reversetranscription–PCR (qPCR; Supplementary Fig. 2F). While GLT-1 was expressed at a relatively low level in bothcontrol and DS astroglia, GLAST was expressed at a higher level in DS astroglia thanthat in control astroglia (P<0.05, n=3–4 fromeach cell line). Interestingly, we found that the DS neurons derived underdirected neuronal differentiation condition showed indistinguishable morphologywith control neurons (Supplementary Fig.2C,G). We used whole-cell patch-clamp recording to measure thespontaneous neuronal activities, and showed that both control and DS neurons at10-week time point fired action potentials and exhibited spontaneouspostsynaptic currents (sPSCs; Supplementary Fig. 2H).


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

Chen C, Jiang P, Xue H, Peterson SE, Tran HT, McCann AE, Parast MM, Li S, Pleasure DE, Laurent LC, Loring JF, Liu Y, Deng W - Nat Commun (2014)

Generation and neural differentiation of DS iPSCs.(a) A schematic procedure for directed and spontaneous differentiationof 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 underdirected and spontaneous differentiation conditions. Scale bars,200 μm. (b,c) Representatives of iPSCsderived from DS patients expressing pluripotent markers Oct4 and SSEA4, as well asNanog and Tra-1-81.(d) Representative of DS iPSC-derived NPCs expressingPax6 andNestin.(e,f) Representatives of βIII-tubulin+ neurons andA2B5+ glial progenitors derived from DS NPCs under directed differentiationconditions. (g) Representatives of astroglia differentiated underdirected astroglial differentiation condition from control (Cont) and DSiPSCs expressing CD44 andvimentin, as well asS100B andGFAP. (h)Representatives of βIII-tubulin+ neurons and S100B+ astroglia derived from DSand Cont NPCs under spontaneous differentiation conditions. Scale bars,50 μm. (i,j) Quantification of pooleddata from Cont and DS lines showing the percentage of βIII-tubulin+ neurons andS100B+ astrogliaderived from DS and Cont NPCs (n=3–5 from each cell line),and the length of the longest neurites of neurons (n=10 from eachcell line) under spontaneous differentiation conditions.Student’s t-test, *P<0.05 and**P<0.01. Blue, 4′,6-diamidino-2-phenylindoledihydrochloride (DAPI)-stained nuclei. Data are presented asmean±s.e.m.
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Related In: Results  -  Collection

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f1: Generation and neural differentiation of DS iPSCs.(a) A schematic procedure for directed and spontaneous differentiationof 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 underdirected and spontaneous differentiation conditions. Scale bars,200 μm. (b,c) Representatives of iPSCsderived from DS patients expressing pluripotent markers Oct4 and SSEA4, as well asNanog and Tra-1-81.(d) Representative of DS iPSC-derived NPCs expressingPax6 andNestin.(e,f) Representatives of βIII-tubulin+ neurons andA2B5+ glial progenitors derived from DS NPCs under directed differentiationconditions. (g) Representatives of astroglia differentiated underdirected astroglial differentiation condition from control (Cont) and DSiPSCs expressing CD44 andvimentin, as well asS100B andGFAP. (h)Representatives of βIII-tubulin+ neurons and S100B+ astroglia derived from DSand Cont NPCs under spontaneous differentiation conditions. Scale bars,50 μm. (i,j) Quantification of pooleddata from Cont and DS lines showing the percentage of βIII-tubulin+ neurons andS100B+ astrogliaderived from DS and Cont NPCs (n=3–5 from each cell line),and the length of the longest neurites of neurons (n=10 from eachcell line) under spontaneous differentiation conditions.Student’s t-test, *P<0.05 and**P<0.01. Blue, 4′,6-diamidino-2-phenylindoledihydrochloride (DAPI)-stained nuclei. Data are presented asmean±s.e.m.
Mentions: To establish an in vitro human cellular model for DS and to investigateneuron-astrocyte interactions, we first generated DS hiPSC lines using thecanonical ‘Yamanaka’ reprogramming method by transducingDS patients’ fibroblasts (Coriell Medical Institute) withretroviruses encoding OCT4,SOX2, KLF4 and c-MYC (Supplementary Fig. 1A). The age-matched hiPSClines from healthy individuals were used as controls. We then differentiated theDS and control hiPSCs to neurons and astroglia via directed or spontaneousdifferentiation procedures shown in Fig. 1a. The hiPSClines expressed pluripotent makers OCT4, SSEA4, NANOG and TRA1-81 (Fig. 1b,c), andwere able to form teratomas that showed structures corresponding to three germlayers (Supplementary Fig. 1B). TheiPSCs and fibroblasts had distinct gene expression pattern, as demonstrated byanalyses of their gene expression profiles (Supplementary Fig. 1C,D). As shown in Supplementary Fig. 1E, thepluripotency of the iPSCs was also evidenced by the results of PluriTest, analgorithm built upon a global gene expression database of a total of 264 PSClines (223 hESC (human embryonic stem cell) and 41 iPSC lines), which has beenused to predict pluripotency accurately and effectively20. Two ofthe iPSC lines generated from DS patients DS1 and DS2 (Supplementary Table 1) maintained a stabletrisomic chromosome 21 karyotype during serial passaging and after neuraldifferentiation (Supplementary Fig.1F), and thus were first used in this study. The control and DS hiPSClines generated NPCs at high efficiency, as indicated by expressing NPC markers,Pax6 and Nestin (Fig. 1dand Supplementary Fig. 2A).Subsequently, under directed neuronal differentiation condition, neuronalprogenitors were further selected and cultured in the presence of neurotrophicfactors brain-derived neurotrophicfactor (BDNF) and glialcell-derived neurotrophic factor (GDNF) (Fig. 1a).Both control and DS hiPSC-derived NPCs were efficiently induced to generateneurons (>85%; Fig. 1e and Supplementary Fig. 2B,C). In parallel, underdirected astroglial differentiation condition by adding bone morphogenetic protein 4(BMP4; Fig.1a)21, the NPCs started to express glial precursormarker A2B5 at early stage (Fig. 1f), and later generatedastroglia after 20 days in culture, as identified by astroglial markersglial fibrillary acidicprotein (GFAP) and S100B (>95%; Fig. 1g and Supplementary Fig. 2D,E). Nearly allthe hiPSC-derived astroglia also expressed CD44, a marker used to identify astrocyte-restrictedprecursor cells, consistent with our recent study on astroglial differentiationof hESCs22, and vimentin, a major cytoskeletal protein expressed in immatureastrocytes23 (Fig. 1g). The robustco-expression of CD44/vimentin and GFAP/S100Bindicated that the majority of hiPSC-derived astroglia were immature, ratherthan mature astrocytes, which better mimic early developmental stages of the DSpathology in the human brain. No significant difference was observed in theefficiency of neuronal and astroglial differentiation between DS and controlhiPSC lines (Supplementary Fig.2B–E) under the directed differentiation conditions. Inaddition, similar to hESC-derived astroglia21, all hiPSCastroglial preparations expressed mRNAs encoding the astrocyte-specificglutamate transporters, glutamate-aspartatetransporter (GLAST) and glutamatetransporter-1 (GLT-1), as detected by quantitative reversetranscription–PCR (qPCR; Supplementary Fig. 2F). While GLT-1 was expressed at a relatively low level in bothcontrol and DS astroglia, GLAST was expressed at a higher level in DS astroglia thanthat in control astroglia (P<0.05, n=3–4 fromeach cell line). Interestingly, we found that the DS neurons derived underdirected neuronal differentiation condition showed indistinguishable morphologywith control neurons (Supplementary Fig.2C,G). We used whole-cell patch-clamp recording to measure thespontaneous neuronal activities, and showed that both control and DS neurons at10-week time point fired action potentials and exhibited spontaneouspostsynaptic currents (sPSCs; Supplementary Fig. 2H).

Bottom Line: DS astroglia exhibit higher levels of reactive oxygen species and lower levels of synaptogenic molecules.Transplantation studies show that DS astroglia do not promote neurogenesis of endogenous neural stem cells in vivo.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.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biochemistry and Molecular Medicine, School of Medicine, University of California, Davis, California 95817, USA [2] Institute for Pediatric Regenerative Medicine, Shriners Hospitals for Children, Sacramento, California 95817, USA [3] Department of Neurology, Institute of Neurology, Tianjin General Hospital, Tianjin Medical University, Tianjin 300070, China [4].

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.

No MeSH data available.


Related in: MedlinePlus