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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

Pathological phenotypes observed in astroglia generated from di- and trisomicDS iPSC lines.(a) Representative images showing that Di-DS3 astroglia and Tri-DS3astroglia express CD44,vimentin,S100B andGFAP, and alsoexhibit disomy and trisomy of HAS21 by fluorescence in situhybridization (FISH) analysis, respectively. (b) qPCR analysis ofS100B,GFAP,TSP-1,iNOS andTSP-2 mRNAexpression in control (Cont) 1 and 2 astroglia, Di-DS3 astroglia and Tri-DS3astroglia with or without the treatment of minocycline. Student’st-test, *P<0.05, **P<0.01, and***P<0.001, comparison between Tri-DS3 Astro with otherindividual group, n=3–4 for each group. (c)Representative images showing NPCs from Di-DS3 and Tri-DS3 iPSCsdifferentiated into βIII-tubulin+ neurons and S100B+ astroglia under spontaneousdifferentiation condition. (d) Representative images showing DS NPCsdifferentiated into βIII-tubulin+ neurons and S100B+ astroglia in the presence orabsence of Di-DS3 ACM or Tri-DS3 ACM. (e) Representative images ofβIII-tubulin+ and activated caspase3+ cells among the groupswith 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-phenylindoledihydrochloride (DAPI)-stained nuclei. (f) Quantification ofβIII-tubulin+ neurons and S100B+ astroglia derived fromDi-DS3 and Tri-DS3 NPCs (n=3–4 from each cell line), andthe length of the longest neurites of neurons (n=10 from each cellline) under spontaneous differentiation condition. (g) Quantificationof βIII-tubulin+ neurons and S100B+ astroglia derived from DSNPCs (n=3–4 from each cell line), and the length of thelongest neurites of neurons (n=10 from each cell line) in thepresence of Di-DS3 ACM or Tri-DS3 ACM. (h) Quantification of pooleddata showing the percentage of βIII-tubulin+ and activated caspase3+ cells among the groupswith 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 activitieswere recorded from Di-DS3 neurons fed with Di-DS3 ACM, Tri-DS3 neurons fedwith Di-DS3 ACM or Tri-DS3-Mino ACM, but not from Di-DS3 neurons and Tri-DS3neurons fed with Tri-DS3 ACM.
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f7: Pathological phenotypes observed in astroglia generated from di- and trisomicDS iPSC lines.(a) Representative images showing that Di-DS3 astroglia and Tri-DS3astroglia express CD44,vimentin,S100B andGFAP, and alsoexhibit disomy and trisomy of HAS21 by fluorescence in situhybridization (FISH) analysis, respectively. (b) qPCR analysis ofS100B,GFAP,TSP-1,iNOS andTSP-2 mRNAexpression in control (Cont) 1 and 2 astroglia, Di-DS3 astroglia and Tri-DS3astroglia with or without the treatment of minocycline. Student’st-test, *P<0.05, **P<0.01, and***P<0.001, comparison between Tri-DS3 Astro with otherindividual group, n=3–4 for each group. (c)Representative images showing NPCs from Di-DS3 and Tri-DS3 iPSCsdifferentiated into βIII-tubulin+ neurons and S100B+ astroglia under spontaneousdifferentiation condition. (d) Representative images showing DS NPCsdifferentiated into βIII-tubulin+ neurons and S100B+ astroglia in the presence orabsence of Di-DS3 ACM or Tri-DS3 ACM. (e) Representative images ofβIII-tubulin+ and activated caspase3+ cells among the groupswith 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-phenylindoledihydrochloride (DAPI)-stained nuclei. (f) Quantification ofβIII-tubulin+ neurons and S100B+ astroglia derived fromDi-DS3 and Tri-DS3 NPCs (n=3–4 from each cell line), andthe length of the longest neurites of neurons (n=10 from each cellline) under spontaneous differentiation condition. (g) Quantificationof βIII-tubulin+ neurons and S100B+ astroglia derived from DSNPCs (n=3–4 from each cell line), and the length of thelongest neurites of neurons (n=10 from each cell line) in thepresence of Di-DS3 ACM or Tri-DS3 ACM. (h) Quantification of pooleddata showing the percentage of βIII-tubulin+ and activated caspase3+ cells among the groupswith 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 activitieswere recorded from Di-DS3 neurons fed with Di-DS3 ACM, Tri-DS3 neurons fedwith Di-DS3 ACM or Tri-DS3-Mino ACM, but not from Di-DS3 neurons and Tri-DS3neurons fed with Tri-DS3 ACM.

Mentions: Previous studies reported the generation of disomic and trisomic subclones eitherfrom the same parental iPSC lines during serial passage47 or fromreprogramming mosaic DS fibroblasts9. In our study, we alsoobserved the loss of one copy of chromosome 21 in one DS iPSC line generatedfrom patient no. DS3. Since we did not observe mosaicism in the fibroblasts frompatient DS3, the disomic subclones might be generated during passaging. Theisogenic disomic and trisomic DS3 iPSCs were isolated by clonal expansion. Wenamed them Di-DS3 (disomy DS3) and Tri-DS3 (trisomy DS3) iPSCs. Like DS1 and DS2iPSCs, Di-DS3 and Tri-DS3 iPSCs were pluripotent cells and had distinct geneexpression profile from the fibroblasts from which they were reprogrammed (Supplementary Fig. 1A–E).The isogenic pair is a perfect tool for us to scrutinize this complex multigenedisorder because the Di-DS3 has the same genetic background as Tri-DS3 exceptfor the extra chromosome 21 (refs 9, 47). To verify the findings from DS 1 and DS2 astroglia,we next differentiated both Di- DS3 and Tri-DS3 iPSCs to astroglia and askedwhether, compared with Di-DS3 astroglia as well as control astroglia, Tri-DS3astroglia would also show the abnormal phenotypes revealed by DS1 and DS2astroglia. As shown in Fig. 7a, astroglia from both Di-DS3and Tri-DS3 iPSCs were identified by the expression of CD44, vimentin, GFAP and S100B, and their copy number of HSA21was verified by fluorescence in situ hybridization analysis. We noticedthat Di-DS3 astroglia appeared smaller in size than Tri-DS3 astroglia (Fig. 7a). However, the size for astroglia from control 1,control 2, DS1 and DS2 iPSCs appeared very similar (Fig.1g). Thus, there seemed no significant correlation between the sizeof astroglia and the DS disease condition. The qPCR results showed that comparedwith Di-DS3 astroglia, Tri-DS3 astroglia expressed much higher levels ofS100B,GFAP andiNOS, andexpressed lower levels of TSP-1 and TSP-2 (Fig. 7b). To furtherconfirm our findings in isogenic lines, we compared the expression of thesegenes in Tri-DS3 astroglia with those of control 1 and 2 astroglia (Fig. 2a). Similar changes of gene expression were observed(Fig. 7b). Consistent with the observation in DS1 andDS2 astroglia, minocyclinetreatment corrected the abnormal expression of these genes in Tri-DS3 astroglia(Fig. 7b). In addition, as shown in Fig. 7c,f, under spontaneous differentiation condition, Tri-DS3 NPCsgave rise to fewer βIII-tubulin+ neurons and more S100B+ astroglia (21.6±3.0%and 76.8±2.4%, respectively), compared with Di-DS3 NPCs(30.9±3.5% and 67.1±2.2%, respectively; P<0.05;n=3–4 from each cell line) and control NPCs(P<0.01; compared with Fig. 1i). Tri-DS3neurons generated under the spontaneous differentiation condition also exhibiteddecreased neurite length compared with Di-DS3 neurons (61.3±4.3 and82.8±3.7 μm for Di-DS3 and Tri-DS3 neurons,respectively; P<0.05; n=10 from each cell line) and controlneurons (P<0.05; compared with Fig. 1j). Asshown in Fig. 7d,g, treating the DS NPCs with Di-DS3 ACM,but not Tri-DS3 ACM restored the reduced neurogenesis of DS NPCs to a levelsimilar to the treatment with control ACM. To further examine the interaction ofDi-DS3 astroglia with neurons, we fed 6- to 7-week-old DS neurons with Di-DS3ACM, Tri-DS3 ACM and Tri-DS3-Mino ACM for 3 days and examined the apoptosis byco-staining of activated caspase-3 and βIII-tubulin. Consistently, as shown in Fig. 7e,h, addition of Tri-DS3 ACM caused more neuronal celldeath compared with the addition of Di-DS3 ACM (the percentage of activecaspase-3+/βIII-tubulin+ neurons was7.5±0.5% and 21.0±1.9% for Di-DS ACM and Tri-DS ACM treatmentgroups, respectively; P<0.01, n=3–6 from eachcell line) and control ACM (P<0.05; compared with Fig. 3e). Addition of Tri-DS3-Mino ACM induced significantly less neuronal cell death thanthe addition of Tri-DS3 ACM (the percentage of active caspase-3+/βIII-tubulin+ neurons was10.4±0.5% for Tri-DS-Mino ACM treatment group; P<0.05,n=3–6). Moreover, we recorded the sPSCs of 6- to 7-week-oldneurons cultured in the presence of different ACM for 3 weeks. As shown in Fig. 7i,j, no synaptic activities were recorded from any ofthe Di-DS3 and Tri-DS3 neurons cultured with Tri-DS3 ACM, while~72.7% (8 of 11) of Di-DS3 neurons fed with Di-DS3 ACM, 69.2% (9 of13) of Tri-DS3 neurons fed with Di-DS3 ACM, and 42.9% (6 of 14) of Tri-DS3neurons fed with Tri-DS3-MinoACM showed synaptic activities.


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)

Pathological phenotypes observed in astroglia generated from di- and trisomicDS iPSC lines.(a) Representative images showing that Di-DS3 astroglia and Tri-DS3astroglia express CD44,vimentin,S100B andGFAP, and alsoexhibit disomy and trisomy of HAS21 by fluorescence in situhybridization (FISH) analysis, respectively. (b) qPCR analysis ofS100B,GFAP,TSP-1,iNOS andTSP-2 mRNAexpression in control (Cont) 1 and 2 astroglia, Di-DS3 astroglia and Tri-DS3astroglia with or without the treatment of minocycline. Student’st-test, *P<0.05, **P<0.01, and***P<0.001, comparison between Tri-DS3 Astro with otherindividual group, n=3–4 for each group. (c)Representative images showing NPCs from Di-DS3 and Tri-DS3 iPSCsdifferentiated into βIII-tubulin+ neurons and S100B+ astroglia under spontaneousdifferentiation condition. (d) Representative images showing DS NPCsdifferentiated into βIII-tubulin+ neurons and S100B+ astroglia in the presence orabsence of Di-DS3 ACM or Tri-DS3 ACM. (e) Representative images ofβIII-tubulin+ and activated caspase3+ cells among the groupswith 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-phenylindoledihydrochloride (DAPI)-stained nuclei. (f) Quantification ofβIII-tubulin+ neurons and S100B+ astroglia derived fromDi-DS3 and Tri-DS3 NPCs (n=3–4 from each cell line), andthe length of the longest neurites of neurons (n=10 from each cellline) under spontaneous differentiation condition. (g) Quantificationof βIII-tubulin+ neurons and S100B+ astroglia derived from DSNPCs (n=3–4 from each cell line), and the length of thelongest neurites of neurons (n=10 from each cell line) in thepresence of Di-DS3 ACM or Tri-DS3 ACM. (h) Quantification of pooleddata showing the percentage of βIII-tubulin+ and activated caspase3+ cells among the groupswith 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 activitieswere recorded from Di-DS3 neurons fed with Di-DS3 ACM, Tri-DS3 neurons fedwith Di-DS3 ACM or Tri-DS3-Mino ACM, but not from Di-DS3 neurons and Tri-DS3neurons fed with Tri-DS3 ACM.
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f7: Pathological phenotypes observed in astroglia generated from di- and trisomicDS iPSC lines.(a) Representative images showing that Di-DS3 astroglia and Tri-DS3astroglia express CD44,vimentin,S100B andGFAP, and alsoexhibit disomy and trisomy of HAS21 by fluorescence in situhybridization (FISH) analysis, respectively. (b) qPCR analysis ofS100B,GFAP,TSP-1,iNOS andTSP-2 mRNAexpression in control (Cont) 1 and 2 astroglia, Di-DS3 astroglia and Tri-DS3astroglia with or without the treatment of minocycline. Student’st-test, *P<0.05, **P<0.01, and***P<0.001, comparison between Tri-DS3 Astro with otherindividual group, n=3–4 for each group. (c)Representative images showing NPCs from Di-DS3 and Tri-DS3 iPSCsdifferentiated into βIII-tubulin+ neurons and S100B+ astroglia under spontaneousdifferentiation condition. (d) Representative images showing DS NPCsdifferentiated into βIII-tubulin+ neurons and S100B+ astroglia in the presence orabsence of Di-DS3 ACM or Tri-DS3 ACM. (e) Representative images ofβIII-tubulin+ and activated caspase3+ cells among the groupswith 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-phenylindoledihydrochloride (DAPI)-stained nuclei. (f) Quantification ofβIII-tubulin+ neurons and S100B+ astroglia derived fromDi-DS3 and Tri-DS3 NPCs (n=3–4 from each cell line), andthe length of the longest neurites of neurons (n=10 from each cellline) under spontaneous differentiation condition. (g) Quantificationof βIII-tubulin+ neurons and S100B+ astroglia derived from DSNPCs (n=3–4 from each cell line), and the length of thelongest neurites of neurons (n=10 from each cell line) in thepresence of Di-DS3 ACM or Tri-DS3 ACM. (h) Quantification of pooleddata showing the percentage of βIII-tubulin+ and activated caspase3+ cells among the groupswith 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 activitieswere recorded from Di-DS3 neurons fed with Di-DS3 ACM, Tri-DS3 neurons fedwith Di-DS3 ACM or Tri-DS3-Mino ACM, but not from Di-DS3 neurons and Tri-DS3neurons fed with Tri-DS3 ACM.
Mentions: Previous studies reported the generation of disomic and trisomic subclones eitherfrom the same parental iPSC lines during serial passage47 or fromreprogramming mosaic DS fibroblasts9. In our study, we alsoobserved the loss of one copy of chromosome 21 in one DS iPSC line generatedfrom patient no. DS3. Since we did not observe mosaicism in the fibroblasts frompatient DS3, the disomic subclones might be generated during passaging. Theisogenic disomic and trisomic DS3 iPSCs were isolated by clonal expansion. Wenamed them Di-DS3 (disomy DS3) and Tri-DS3 (trisomy DS3) iPSCs. Like DS1 and DS2iPSCs, Di-DS3 and Tri-DS3 iPSCs were pluripotent cells and had distinct geneexpression profile from the fibroblasts from which they were reprogrammed (Supplementary Fig. 1A–E).The isogenic pair is a perfect tool for us to scrutinize this complex multigenedisorder because the Di-DS3 has the same genetic background as Tri-DS3 exceptfor the extra chromosome 21 (refs 9, 47). To verify the findings from DS 1 and DS2 astroglia,we next differentiated both Di- DS3 and Tri-DS3 iPSCs to astroglia and askedwhether, compared with Di-DS3 astroglia as well as control astroglia, Tri-DS3astroglia would also show the abnormal phenotypes revealed by DS1 and DS2astroglia. As shown in Fig. 7a, astroglia from both Di-DS3and Tri-DS3 iPSCs were identified by the expression of CD44, vimentin, GFAP and S100B, and their copy number of HSA21was verified by fluorescence in situ hybridization analysis. We noticedthat Di-DS3 astroglia appeared smaller in size than Tri-DS3 astroglia (Fig. 7a). However, the size for astroglia from control 1,control 2, DS1 and DS2 iPSCs appeared very similar (Fig.1g). Thus, there seemed no significant correlation between the sizeof astroglia and the DS disease condition. The qPCR results showed that comparedwith Di-DS3 astroglia, Tri-DS3 astroglia expressed much higher levels ofS100B,GFAP andiNOS, andexpressed lower levels of TSP-1 and TSP-2 (Fig. 7b). To furtherconfirm our findings in isogenic lines, we compared the expression of thesegenes in Tri-DS3 astroglia with those of control 1 and 2 astroglia (Fig. 2a). Similar changes of gene expression were observed(Fig. 7b). Consistent with the observation in DS1 andDS2 astroglia, minocyclinetreatment corrected the abnormal expression of these genes in Tri-DS3 astroglia(Fig. 7b). In addition, as shown in Fig. 7c,f, under spontaneous differentiation condition, Tri-DS3 NPCsgave rise to fewer βIII-tubulin+ neurons and more S100B+ astroglia (21.6±3.0%and 76.8±2.4%, respectively), compared with Di-DS3 NPCs(30.9±3.5% and 67.1±2.2%, respectively; P<0.05;n=3–4 from each cell line) and control NPCs(P<0.01; compared with Fig. 1i). Tri-DS3neurons generated under the spontaneous differentiation condition also exhibiteddecreased neurite length compared with Di-DS3 neurons (61.3±4.3 and82.8±3.7 μm for Di-DS3 and Tri-DS3 neurons,respectively; P<0.05; n=10 from each cell line) and controlneurons (P<0.05; compared with Fig. 1j). Asshown in Fig. 7d,g, treating the DS NPCs with Di-DS3 ACM,but not Tri-DS3 ACM restored the reduced neurogenesis of DS NPCs to a levelsimilar to the treatment with control ACM. To further examine the interaction ofDi-DS3 astroglia with neurons, we fed 6- to 7-week-old DS neurons with Di-DS3ACM, Tri-DS3 ACM and Tri-DS3-Mino ACM for 3 days and examined the apoptosis byco-staining of activated caspase-3 and βIII-tubulin. Consistently, as shown in Fig. 7e,h, addition of Tri-DS3 ACM caused more neuronal celldeath compared with the addition of Di-DS3 ACM (the percentage of activecaspase-3+/βIII-tubulin+ neurons was7.5±0.5% and 21.0±1.9% for Di-DS ACM and Tri-DS ACM treatmentgroups, respectively; P<0.01, n=3–6 from eachcell line) and control ACM (P<0.05; compared with Fig. 3e). Addition of Tri-DS3-Mino ACM induced significantly less neuronal cell death thanthe addition of Tri-DS3 ACM (the percentage of active caspase-3+/βIII-tubulin+ neurons was10.4±0.5% for Tri-DS-Mino ACM treatment group; P<0.05,n=3–6). Moreover, we recorded the sPSCs of 6- to 7-week-oldneurons cultured in the presence of different ACM for 3 weeks. As shown in Fig. 7i,j, no synaptic activities were recorded from any ofthe Di-DS3 and Tri-DS3 neurons cultured with Tri-DS3 ACM, while~72.7% (8 of 11) of Di-DS3 neurons fed with Di-DS3 ACM, 69.2% (9 of13) of Tri-DS3 neurons fed with Di-DS3 ACM, and 42.9% (6 of 14) of Tri-DS3neurons fed with Tri-DS3-MinoACM showed synaptic activities.

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