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Modeling Alexander disease with patient iPSCs reveals cellular and molecular pathology of astrocytes.

Kondo T, Funayama M, Miyake M, Tsukita K, Era T, Osaka H, Ayaki T, Takahashi R, Inoue H - Acta Neuropathol Commun (2016)

Bottom Line: Alexander disease is a fatal neurological illness characterized by white-matter degeneration and formation of Rosenthal fibers, which contain glial fibrillary acidic protein as astrocytic inclusion.We established induced pluripotent stem cells from Alexander disease patients, and differentiated induced pluripotent stem cells into astrocytes.Alexander disease patient astrocytes exhibited Rosenthal fiber-like structures, a key Alexander disease pathology, and increased inflammatory cytokine release compared to healthy control.

View Article: PubMed Central - PubMed

Affiliation: Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan.

ABSTRACT
Alexander disease is a fatal neurological illness characterized by white-matter degeneration and formation of Rosenthal fibers, which contain glial fibrillary acidic protein as astrocytic inclusion. Alexander disease is mainly caused by a gene mutation encoding glial fibrillary acidic protein, although the underlying pathomechanism remains unclear. We established induced pluripotent stem cells from Alexander disease patients, and differentiated induced pluripotent stem cells into astrocytes. Alexander disease patient astrocytes exhibited Rosenthal fiber-like structures, a key Alexander disease pathology, and increased inflammatory cytokine release compared to healthy control. These results suggested that Alexander disease astrocytes contribute to leukodystrophy and a variety of symptoms as an inflammatory source in the Alexander disease patient brain. Astrocytes, differentiated from induced pluripotent stem cells of Alexander disease, could be a cellular model for future translational medicine.

No MeSH data available.


Related in: MedlinePlus

iPSCs from AxD patients and healthy controls could differentiate into astrocytes with high purity. a Schematic procedures for astroglial differentiation. b Differentiated iPSCs at day 24 expressed neural stem cell markers NESTIN and GFAP. Neural cells at day 60 expressed neuronal or astrocytic marker TUJ1 or GFAP. Most enriched astrocytes expressed GFAP. Scale bars = 20 μm. c Estimation of astroglial differentiation from control and AxD iPSCs. After 180 days of differentiation, astrocytes were immunostained with an antibody against S100β (red color). Scale bars = 20 μm. d Calculated purity of astrocytic differentiation Data represent mean ± SD (biological replicates, n = 3 from randomly picked fields per clone). Two-way analysis of variance (ANOVA) did not show significant variation. F (5, 12) =0.2432; p = 0.935
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Fig2: iPSCs from AxD patients and healthy controls could differentiate into astrocytes with high purity. a Schematic procedures for astroglial differentiation. b Differentiated iPSCs at day 24 expressed neural stem cell markers NESTIN and GFAP. Neural cells at day 60 expressed neuronal or astrocytic marker TUJ1 or GFAP. Most enriched astrocytes expressed GFAP. Scale bars = 20 μm. c Estimation of astroglial differentiation from control and AxD iPSCs. After 180 days of differentiation, astrocytes were immunostained with an antibody against S100β (red color). Scale bars = 20 μm. d Calculated purity of astrocytic differentiation Data represent mean ± SD (biological replicates, n = 3 from randomly picked fields per clone). Two-way analysis of variance (ANOVA) did not show significant variation. F (5, 12) =0.2432; p = 0.935

Mentions: The astrocytic differentiation protocol for human iPSCs was modified from our previous method [12] (Fig. 2a). In the neural patterning stage, differentiated cells expressed NESTIN (marker of neural stem cells) or GFAP (marker of radial glia in cortical development) (Fig. 2b). After 2 months, differentiated cells abundantly expressed TUJ1 (neuronal marker) (Fig. 2b). By repeating low-density passage, differentiated neurons, without proliferation, failed to attach to the dish and were selectively removed. After five passages and more than 6 months of cultivation, iPSC-derived astrocytes were enriched (Fig. 2b). Differentiated astrocytes abundantly expressed S100β (Fig. 2c) and GFAP (Fig. 3a and b), which are commonly used as astrocytes markers. We did not observe any obvious difference in astrocytic differentiation efficacy among all individuals (Fig. 2d).Fig. 2


Modeling Alexander disease with patient iPSCs reveals cellular and molecular pathology of astrocytes.

Kondo T, Funayama M, Miyake M, Tsukita K, Era T, Osaka H, Ayaki T, Takahashi R, Inoue H - Acta Neuropathol Commun (2016)

iPSCs from AxD patients and healthy controls could differentiate into astrocytes with high purity. a Schematic procedures for astroglial differentiation. b Differentiated iPSCs at day 24 expressed neural stem cell markers NESTIN and GFAP. Neural cells at day 60 expressed neuronal or astrocytic marker TUJ1 or GFAP. Most enriched astrocytes expressed GFAP. Scale bars = 20 μm. c Estimation of astroglial differentiation from control and AxD iPSCs. After 180 days of differentiation, astrocytes were immunostained with an antibody against S100β (red color). Scale bars = 20 μm. d Calculated purity of astrocytic differentiation Data represent mean ± SD (biological replicates, n = 3 from randomly picked fields per clone). Two-way analysis of variance (ANOVA) did not show significant variation. F (5, 12) =0.2432; p = 0.935
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Fig2: iPSCs from AxD patients and healthy controls could differentiate into astrocytes with high purity. a Schematic procedures for astroglial differentiation. b Differentiated iPSCs at day 24 expressed neural stem cell markers NESTIN and GFAP. Neural cells at day 60 expressed neuronal or astrocytic marker TUJ1 or GFAP. Most enriched astrocytes expressed GFAP. Scale bars = 20 μm. c Estimation of astroglial differentiation from control and AxD iPSCs. After 180 days of differentiation, astrocytes were immunostained with an antibody against S100β (red color). Scale bars = 20 μm. d Calculated purity of astrocytic differentiation Data represent mean ± SD (biological replicates, n = 3 from randomly picked fields per clone). Two-way analysis of variance (ANOVA) did not show significant variation. F (5, 12) =0.2432; p = 0.935
Mentions: The astrocytic differentiation protocol for human iPSCs was modified from our previous method [12] (Fig. 2a). In the neural patterning stage, differentiated cells expressed NESTIN (marker of neural stem cells) or GFAP (marker of radial glia in cortical development) (Fig. 2b). After 2 months, differentiated cells abundantly expressed TUJ1 (neuronal marker) (Fig. 2b). By repeating low-density passage, differentiated neurons, without proliferation, failed to attach to the dish and were selectively removed. After five passages and more than 6 months of cultivation, iPSC-derived astrocytes were enriched (Fig. 2b). Differentiated astrocytes abundantly expressed S100β (Fig. 2c) and GFAP (Fig. 3a and b), which are commonly used as astrocytes markers. We did not observe any obvious difference in astrocytic differentiation efficacy among all individuals (Fig. 2d).Fig. 2

Bottom Line: Alexander disease is a fatal neurological illness characterized by white-matter degeneration and formation of Rosenthal fibers, which contain glial fibrillary acidic protein as astrocytic inclusion.We established induced pluripotent stem cells from Alexander disease patients, and differentiated induced pluripotent stem cells into astrocytes.Alexander disease patient astrocytes exhibited Rosenthal fiber-like structures, a key Alexander disease pathology, and increased inflammatory cytokine release compared to healthy control.

View Article: PubMed Central - PubMed

Affiliation: Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan.

ABSTRACT
Alexander disease is a fatal neurological illness characterized by white-matter degeneration and formation of Rosenthal fibers, which contain glial fibrillary acidic protein as astrocytic inclusion. Alexander disease is mainly caused by a gene mutation encoding glial fibrillary acidic protein, although the underlying pathomechanism remains unclear. We established induced pluripotent stem cells from Alexander disease patients, and differentiated induced pluripotent stem cells into astrocytes. Alexander disease patient astrocytes exhibited Rosenthal fiber-like structures, a key Alexander disease pathology, and increased inflammatory cytokine release compared to healthy control. These results suggested that Alexander disease astrocytes contribute to leukodystrophy and a variety of symptoms as an inflammatory source in the Alexander disease patient brain. Astrocytes, differentiated from induced pluripotent stem cells of Alexander disease, could be a cellular model for future translational medicine.

No MeSH data available.


Related in: MedlinePlus