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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

Astrocytes of Alexander disease showed GFAP-positive aggregates. a Most iPSC-derived astrocytes showed GFAP-positive staining (green color). In healthy control, GFAP showed filamentous structure. In Alexander disease, GFAP mainly showed filamentous structure, but also fibrous/amorphous (arrows) or dot-like (arrowheads) aggregates. Scale bars = 5 μm. b Calculated purity of astrocytic differentiation data represent mean ± SD (biological replicates, n = 3 from randomly picked fields per clone). c Calculated positivity of GFAP aggregates. Data represent mean ± SD (biological replicates, n = 3 from randomly picked fields per clone)
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Fig3: Astrocytes of Alexander disease showed GFAP-positive aggregates. a Most iPSC-derived astrocytes showed GFAP-positive staining (green color). In healthy control, GFAP showed filamentous structure. In Alexander disease, GFAP mainly showed filamentous structure, but also fibrous/amorphous (arrows) or dot-like (arrowheads) aggregates. Scale bars = 5 μm. b Calculated purity of astrocytic differentiation data represent mean ± SD (biological replicates, n = 3 from randomly picked fields per clone). c Calculated positivity of GFAP aggregates. Data represent mean ± SD (biological replicates, n = 3 from randomly picked fields per clone)

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)

Astrocytes of Alexander disease showed GFAP-positive aggregates. a Most iPSC-derived astrocytes showed GFAP-positive staining (green color). In healthy control, GFAP showed filamentous structure. In Alexander disease, GFAP mainly showed filamentous structure, but also fibrous/amorphous (arrows) or dot-like (arrowheads) aggregates. Scale bars = 5 μm. b Calculated purity of astrocytic differentiation data represent mean ± SD (biological replicates, n = 3 from randomly picked fields per clone). c Calculated positivity of GFAP aggregates. Data represent mean ± SD (biological replicates, n = 3 from randomly picked fields per clone)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4940830&req=5

Fig3: Astrocytes of Alexander disease showed GFAP-positive aggregates. a Most iPSC-derived astrocytes showed GFAP-positive staining (green color). In healthy control, GFAP showed filamentous structure. In Alexander disease, GFAP mainly showed filamentous structure, but also fibrous/amorphous (arrows) or dot-like (arrowheads) aggregates. Scale bars = 5 μm. b Calculated purity of astrocytic differentiation data represent mean ± SD (biological replicates, n = 3 from randomly picked fields per clone). c Calculated positivity of GFAP aggregates. Data represent mean ± SD (biological replicates, n = 3 from randomly picked fields per clone)
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